U.S. patent number 5,512,490 [Application Number 08/289,001] was granted by the patent office on 1996-04-30 for optical sensor, optical sensing apparatus, and methods for detecting an analyte of interest using spectral recognition patterns.
This patent grant is currently assigned to Trustees of Tufts College. Invention is credited to John S. Kauer, David R. Walt.
United States Patent |
5,512,490 |
Walt , et al. |
April 30, 1996 |
**Please see images for:
( Certificate of Correction ) ** |
Optical sensor, optical sensing apparatus, and methods for
detecting an analyte of interest using spectral recognition
patterns
Abstract
The present invention is an optical detection and identification
system and provides an optic sensor, an optic sensing apparatus and
methodology for detecting and evaluating one or more analytes or
ligands of interest, either alone or in admixture. The optic sensor
of the system is comprised of a supporting member and an array
formed of heterogeneous, semi-selective thin films which function
as sensing receptor units and are able to detect a variety of
different analytes and ligands using spectral recognition
patterns.
Inventors: |
Walt; David R. (Lexington,
MA), Kauer; John S. (Weston, MA) |
Assignee: |
Trustees of Tufts College
(Medford, MA)
|
Family
ID: |
23109582 |
Appl.
No.: |
08/289,001 |
Filed: |
August 11, 1994 |
Current U.S.
Class: |
436/171;
250/459.1; 356/317; 422/82.05; 422/91 |
Current CPC
Class: |
G01N
21/6428 (20130101); G01N 21/6452 (20130101); G01N
21/77 (20130101); G01N 2021/6484 (20130101); G01N
2021/7793 (20130101) |
Current International
Class: |
G01N
21/64 (20060101); G01N 21/77 (20060101); G01N
021/77 () |
Field of
Search: |
;422/82.06,82.07,82.05,82.08,86,91 ;436/171,172,164
;250/458.1,271,461.1,461.2,459.1 ;356/317,318,417 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Olness, D. et al, Report (1984) UCID-20047 Chem. Abstracts AN
1986:490265 (Abstract Only). .
Smardzewski, Talanta (1988), 35(2) 95-101 Abstract Only. .
Yokoyama, K. & F. Ebisawa, Anal. Chem. 65:673-677 (1993). .
Patrash, S. J. & E. T. Zellers, Anal. Chem. 65:2055-2066
(1993). .
Grate et al., Anal. Chem. 65:1868-1881 (1993)..
|
Primary Examiner: Snay; Jeffrey R.
Attorney, Agent or Firm: Prashker; David
Claims
What we claim is:
1. An optical sensor for detecting an analyte of interest in a
fluid sample, said optical sensor comprising:
a supporting member; and
an optic array formed of multiple semi-selective sensing receptor
units which differ in their constituent chemical formulations,
which differ in their spectral characteristics, which are
immobilized at different spatial positions on said supporting
member for reactive contact with the fluid sample, and which react
concurrently and semi-selectively but spectrally differently with
an individual analyte of interest, each of said multiple
semi-selective sensing receptor units of said optic array being
comprised of
a polymeric substance of predetermined chemical composition,
and
a semi-selective dye compound of predetermined chemical composition
which has characteristic spectral properties, is disposed in
admixture with said polymeric substance, and can react
semi-selectively and spectrally differently over time with more
than one analyte,
(a) wherein said admixed dye compound absorbs light energy of a
predetermined wavelength and, in the presence of said polymeric
substance without an analyte able to react semi-selectively, yields
a baseline spectral response over time which is optically
detectable and recognizable as showing an absence of analyte,
and
(b) wherein said admixed dye compound absorbs light energy of a
predetermined wavelength and, in the presence of said polymeric
substance and at least one analyte of interest able to react
semi-selectively, generates a modified spectral response over time
which is optically detectable and recognizable as showing the
spectral consequence of semi-selective reaction with the analyte of
interest,
said multiple semi-selective sensing receptor units of said optic
array presenting a plurality of differing and alternative modified
spectral responses after concurrent semi-selective reaction with
the analyte of interest in the fluid sample, the spectral pattern
formed collectively by said plurality of differing and alternative
modified spectral responses resulting in spectral recognition
progression pattern means by which to detect and identify that
analyte of interest.
2. An optical sensor for detecting a first analyte of interest
which is intermixed with at least one other analyte of interest in
a fluid sample, said optical sensor comprising:
a supporting member; and
an optic array formed of multiple semi-selective sensing receptor
units which differ in their constituent chemical formulations,
which differ in their spectral characteristics, which are
immobilized at different spatial positions on said supporting
member for reactive contact with the fluid sample, and which react
concurrently and semi-selectively but spectrally differently with
an individual analyte of interest, each of said multiple
semi-selective sensing receptor units of said optic, array being
comprised of
a polymeric substance of predetermined chemical composition,
and
a semi-selective dye compound of predetermined chemical composition
which has characteristic spectral properties, is disposed in
admixture with said polymeric substance, and can react
semi-selectively and spectrally differently over time with more
than one analyte,
(a) wherein said admixed dye compound absorbs light energy of a
predetermined wavelength and, in the presence of said polymeric
substance without an analyte able to react semi-selectively, yields
a baseline spectral response which is optically detectable and
recognizable as showing an absence of analyte, and
(b) wherein said admixed dye compound absorbs light energy of a
predetermined wavelength and, in the presence of said polymeric
substance and a first analyte of interest able to react
semi-selectively, generates a first modified spectral response over
time which is optically detectable and recognizable as showing the
spectral consequence of semi-selective reaction with the first
analyte of interest, and
(c) wherein said admixed dye compound absorbs light energy of a
predetermined wavelength and, in the presence of said polymeric
substance and at least a second analyte of interest able to react
semi-selectively, generates a second modified spectral response
which is optically detectable and recognizable as showing the
spectral consequence of semi-selective reaction with the second
analyte of interest,
said multiple semi-selective sensing receptor units of said optic
array presenting a plurality of differing and alternative modified
spectral responses after semi-selective reaction with each of said
first and second analytes of interest, the spectral pattern formed
collectively by said plurality of differing and alternative
modified spectral responses for each of said first and second
analytes of interest resulting in individual spectral recognition
progression pattern means by which to detect and identify each of
the analytes of interest in the fluid sample.
3. An optical sensing apparatus for detecting an analyte of
interest in a fluid sample, said optical sensing apparatus
comprising:
a supporting member; and
an optic array formed of multiple semi-selective sensing receptor
units which differ in their constituent chemical formulations,
which differ in their spectral characteristics, which are
immobilized at different spatial positions on said supporting
member for reactive contact with the fluid sample, and which react
concurrently and semi-selectively but spectrally differently with
an individual analyte of interest, each of said multiple
semi-selective sensing receptor units of said optic array being
comprised of
a polymeric substance of predetermined chemical composition,
and
a semi-selective dye compound of predetermined chemical composition
which has characteristic spectral properties, is disposed in
admixture with said polymeric substance, and can react
semi-selectively and spectrally differently with more than one
analyte,
(a) wherein said admixed dye compound absorbs light energy of a
predetermined wavelength and, in the presence of said polymeric
substance without an analyte able to react semi-selectively, yields
a baseline spectral response over time which is optically
detectable and recognizable as showing an absence of analyte,
and
(b) wherein said admixed dye compound absorbs light energy of a
predetermined wavelength and, in the presence of said polymeric
substance and at least one analyte of interest able to react
semi-selectively, generates a modified spectral response over time
which is optically detectable and recognizable as showing the
spectral consequence of semi-selective reaction with the analyte of
interest,
said multiple semi-selective sensing receptor units of said optic
array presenting a plurality of differing and alternative modified
spectral responses after concurrent semi-selective reaction with
the analyte of interest in the fluid sample, the spectral pattern
formed collectively by said plurality of differing and alternative
modified spectral responses for an analyte of interest resulting in
spectral recognition progression pattern means by which to detect
and identify that analyte of interest;
means for introducing a fluid sample to said optic array for
semi-selective reactive contact;
means for introducing light energy of a predetermined wavelength to
said multiple semi-selective sensing receptor units of said optic
array; and
computerized optical detection and evaluation means for optically
detecting said plurality of differing and alternative modified
spectral responses generated by said semi-selective sensing
receptor units and for evaluating said resulting spectral
recognition progression pattern means to determine the presence of
that analyte of interest in the fluid sample.
4. An optical sensing apparatus for detecting a first analyte of
interest which is intermixed with at least one other analyte of
interest in a fluid sample, said optical sensing apparatus
comprising:
a supporting member; and
an optic array formed of multiple semi-selective sensing receptor
units which differ in their constituent chemical formulations,
which differ in their spectral characteristics, which are
immobilized at different spatial positions on said supporting
member for reactive contact with the fluid sample, and which react
concurrently and semi-selectively but spectrally differently with
an individual analyte of interest, each of said multiple
semi-selective sensing receptor units of said optic array being
comprised of
a polymeric substance of predetermined chemical composition,
and
a semi-selective dye compound of predetermined chemical composition
which has characteristic spectral properties, is disposed in
admixture with said polymeric substance, and can react
semi-selectively and spectrally differently over time with more
than one analyte,
(a) wherein said admixed dye compound absorbs light energy of a
predetermined wavelength and, in the presence of said polymeric
substance without an analyte able to react semi-selectively, yields
a baseline spectral response over time which is optically
detectable and recognizable as showing an absence of analyte,
and
(b) wherein said admixed dye compound absorbs light energy of a
predetermined wavelength and, in the presence of said polymeric
substance and a first analyte of interest able to react
semi-selectively, generates a modified spectral response over time
which is optically detectable and recognizable as showing the
spectral consequence of semi-selective reaction with the analyte of
interest, and
(c) wherein said admixed dye compound absorbs light energy of a
predetermined wavelength and, in the presence of said polymeric
substance and at least a second analyte of interest able to react
semi-selectively, generates a second modified spectral response
over time which is optically detectable and recognizable as showing
the spectral consequence of semi-selective reaction with the second
analyte of interest,
said multiple semi-selective sensing receptor units of said optic
array presenting a plurality of differing and alternative modified
spectral responses after concurrent semi-selective reaction with
each of the first and second analytes of interest, the spectral
pattern formed collectively by said plurality of differing and
alternative modified spectral responses for each of the first and
second analytes of interest resulting in individual spectral
recognition progression pattern means by which to detect and
identify each of the analytes of interest in the fluid sample;
means for introducing a fluid sample to said optic array for
semi-selective reactive contact;
means for introducing light energy of a predetermined wavelength to
said multiple semi-selective sensing receptor units of said optic
array; and
computerized optical detection and evaluation means for optically
detecting said plurality of differing and alternative modified
spectral responses generated by said semi-selective sensing
receptor units and for evaluating said resulting spectral
recognition progression pattern means individually to determine the
presence of each of the analytes of interest in the fluid
sample.
5. An optical method for detecting an analyte of interest in a
fluid sample, said optical method comprising the steps of:
providing an optical sensor comprised of
a supporting member; and
an optic array formed of multiple semi-selective sensing receptor
units which differ in their constituent chemical formulations,
which differ in their spectral characteristics, which are
immobilized at different spatial positions on said supporting
member for reactive contact with the fluid sample, and which react
concurrently and semi-selectively but spectrally differently with
an individual analyte of interest, each of said multiple
semi-selective sensing receptor units of said optic array being
comprised of
(a) a polymeric substance of predetermined chemical composition,
and
(b) a semi-selective dye compound of predetermined chemical
composition which has characteristic spectral properties, is
disposed in admixture with said polymeric substance, and can react
semi-selectively and spectrally differently over time with more
than one analyte,
(i) wherein said admixed dye compound absorbs light energy of a
predetermined wavelength and, in the presence of said polymeric
substance without an analyte able to react semi-selectively, yields
a baseline spectral response progression over time which is
optically detectable and recognizable as showing an absence of
analyte, and
(ii) wherein said admixed dye compound absorbs light energy of
predetermined wavelength and, in the presence of said polymeric
substance and an analyte of interest able to react
semi-selectively, generates a modified spectral response over time
which is optically detectable and recognizable as showing the
spectral consequence of semi-selective reaction with the analyte of
interest,
said multiple semi-selective sensing receptor units of said optic
array presenting a plurality of differing and alternative modified
spectral responses after concurrent semi-selective reaction with
the analyte of interest, the spectral pattern formed collectively
by said plurality of differing and alternative modified spectral
responses resulting in spectral recognition progression pattern
means by which to detect and identify that analyte of interest;
introducing the fluid sample to said optical sensor for
semi-selective reactive contact;
introducing light energy of a predetermined wavelength to said
multiple semi-selective sensing receptor units of said optical
sensor;
optically detecting said plurality of differing and alternative
modified spectral responses generated over time by said
semi-selective sensing receptor units of said optical sensor
collectively to form said resulting spectral recognition
progression pattern means; and
evaluating said resulting spectral recognition progression pattern
means using computerized means to determine the presence of that
analyte of interest in the fluid sample.
6. An optical method for detecting a first analyte of interest
which is intermixed with at least one other analyte of interest in
a fluid sample, said method comprising the steps of:
providing an optical sensor comprising:
a supporting member; and
an optic array formed of multiple semi-selective sensing receptor
units which differ in their constituent chemical formulations,
which differ in their spectral characteristics, which are
immobilized at different spatial positions on said supporting
member for reactive contact with the fluid sample, and which react
concurrently and semi-selectively but spectrally differently with
an individual analyte of interest, each of said multiple
semi-selective sensing receptor units of said optic array being
comprised of
(a) a polymeric substance of predetermined chemical composition,
and
(b) a semi-selective dye compound of predetermined chemical
composition which has characteristic spectral properties, is
disposed in admixture with said polymeric substance, and can react
semi-selectively and spectrally differently over time with more
than one analyte,
(i) wherein said admixed dye compound absorbs light energy of a
predetermined wavelength and, in the presence of said polymeric
substance without an analyte able to react semi-selectively, yields
a baseline spectral response over time which is optically
detectable and recognizable as showing an absence of analyte,
and
(ii) wherein said admixed dye compound absorbs light energy of a
predetermined wavelength and, in the presence of said polymeric
substance and a first analyte of interest able to react
semi-selectively, generates a first modified spectral response over
time which is optically detectable and recognizable as showing the
spectral consequence of semi-selective reaction with the first
analyte of interest, and
(iii) wherein said admixed dye compound absorbs light energy of a
predetermined wavelength and, in the presence of said polymeric
substance and at least a second analyte of interest able to react
semi-selectively, generates a second modified spectral response
over time which is optically detectable and recognizable as showing
the spectral consequence of semi-selective reaction with a second
analyte of interest,
said multiple semi-selective sensing receptor units of said optic
array presenting a plurality of differing and alternative modified
spectral responses after concurrent semi-selective reaction with
each of the first and second analytes of interest, the spectral
pattern formed collectively by said plurality of differing and
alternative modified spectral responses for each of the first and
second analytes of interest resulting in individual spectral
recognition progression pattern means by which to detect and
identify each of the analytes of interest;
introducing the fluid sample to said optical sensor for
semi-selective reactive contact;
introducing light energy of a predetermined wavelength to said
multiple semi-selective sensing receptor units of said optical
sensor;
optically detecting said plurality of differing and alternative
modified spectral responses generated by said semi-selective
sensing receptor units of said optical sensor collectively to form
individual resulting spectral recognition progression pattern
means; and
evaluating said resulting spectral recognition progression pattern
means individually using computerized means to determine the
presence of each of the analytes of interest in the fluid sample.
Description
FIELD OF THE INVENTION
The present invention is concerned with optical chemical sensors
and sensing apparatus for the detection of gaseous and liquid
analytes in a manner analogous to the mammalian olfactory system;
and is particularly directed to the use of pattern recognition
techniques for detection and evaluation of optical data generated
by an array of thin film, semi-selective sensing receptor
units.
BACKGROUND OF THE INVENTION
In the last ten to fifteen years, intensive efforts and
developments have occurred in chemical sensor research and in
chemical sensing detection methods and instruments for occupational
safety, environmental monitoring, and for processing or quality
control. Optical sensors and sensing apparatus have been of
particular interest; and the use of optical fibers and optical
fiber strands in combination with light energy absorbing dyes for
medical, biochemical, and chemical analytical determinations has
undergone rapid development.
Conventional optical sensors and optical sensing apparatus, whether
or not optical fibers are used, typically employ one or more light
energy absorbing dyes which are specific for an analyte of interest
and will selectively bind with that analyte. Thus, when light of an
appropriate wavelength is introduced to and has been absorbed by
the dye, the light energy which is either not absorbed or is
returned as an emission is observed and measured by a detection
system. The interactions between the light energy conveyed and the
properties of the specifically--binding, light absorbing dye--in
the presence of one or more ligands or analytes of interest and in
the absence of any ligands or analytes whatsoever-provide an
optical basis for both qualitative and quantitative determinations.
This traditional approach, for both optical and non-optical sensors
alike, has therefore been to create highly selective sensors by
finding and using specific binding materials. This overall approach
consequently results in creating one sensor for each analyte or
ligand of interest to be detected. The one analyte/one sensor
approach thus has been previously and remains today the overriding
guiding principle and axiom for optical chemical sensors and
optical chemical sensing apparatus.
It is useful to recognize and appreciate the stringent demands and
essential requirements of the traditional one analyte/one sensor
approach. These include: (1) each sensor must employ and use one
highly selective/specific binding agent for binding and reaction
with a single analyte or ligand of interest in a sample; (2) the
sensor relies and depends upon the energy signal generated by the
selective binding agent as the means for detecting and determining
the presence of the single analyte or ligand in the sample; (3) the
approach requires that for detection of multiple analytes or
ligands, a series of different selective binding agents with
individual and different binding specificities are used together as
multiples concurrently or in sequence; and (4) the specific binding
and signal generation of the sensor can be accomplished using a
variety of different binding agents including colorimetric or
fluorescent dyes, selective polymer films, or biological receptors
such as enzymes and antibodies. In each instance, one sensor must
be created for the detection of each analyte or ligand of
interest.
In comparison, it will be noted that nature has created a
biological sensing system which is markedly different both in
structure and function from the man-made traditional chemical
sensor approach. For example, the mammalian olfactory system is an
in-vivo sensor for vaporous odors which is not matched by any
artificially synthesized sensor to date in detection limit and
discriminatory power. Vapor odor reception is an interaction
between olfactory receptor cells and the vapor molecules. In short,
the odor is "sensed" by sensory neurons in the olfactory
epithelium, followed by the formation of a neuronal activity
pattern which consists from multiple different responses of
receptor cells to the one odor. The activity pattern of affected
sensory neurons is projected to the olfactory bulb; and the
response patterns are then transmitted to the other various brain
regions for recognition and identification. This system is unique
because, rather than having one receptor for each specific
molecule, a variety of different sensory neurons are involved; and
each of them recognizes one or more properties of the odor. As a
result, a large population of different sensory neurons will
respond to a given odor; but each neuron responds
differently--thereby giving rise to an odor-specific output
response pattern. It is believed that the neuronal circuitry of the
olfactory bulb recognizes and identifies this odor-specific output
pattern through processing with its circuits.
The concept of employing chemical sensors using pattern recognition
systems analogous to those of the mammalian olfactory system has
been modestly explored by a number of different research
laboratories; and the few detection systems using such an analogous
pattern recognition approach today are popularly referred to as
"smart sensor systems", or "odor-sensing systems", or "electronic
noses". Representative of these research investigations and systems
are the following publications: Abe et al., Anal. Chem. Acta.
194:1-9 (1987) and 215:155-168 (1988); Carey et al., Anal. chem.
58:149-153 (1986), Anal. Chem. 58:3077-3084 (1986), Sens. Actuators
9:223-224 (1986), Anal. Chem. 59:1529-1534 (1987); Ema et al.,
Sens. Actuators 18:291-296 (1989); Abe et el., Anal. Chem. Acta.
215:155-168 (1988); Stetter et al., Anal. Chem. 58:860-866 (1986);
Gardner, J. W., Sens. Actuators B 4:109-115 (1991); Muller, R. and
E. Lang, Sens. Actuators 9:39-48 (1986); Muller, R., Sens.
Actuators B 4:35-39 (1991); Ballantine et el., Anal. Chem.
58:3058-3066 (1986); Rose-Pehrrson et el., Anal. Chem. 60:2801-2811
(1988); and Grate et al., Sens. Actuators B 3:85-111 (1991).
The use of chemical sensors with pattern recognition capabilities
have to date taken two non-optical structural formats: the use of
surface acoustic wave (SAW) or bulk acoustic wave (BAW) sensors;
and the use of piezoelectric sensors. The bulk acoustic wave
sensors and surface acoustic wave devices are piezoelectric
crystals which have been coated on the external surface with a
polymer or a high boiling liquid. BAW and SAW devices are chemical
sensors which rely on mass changes, oscillator circuitry and
electronic controls to operate the various subsystems and to
collect and process the data received. Such detection systems are
well described by the following publications: Grate et el., Anal.
Chem. 65:1868-1881 (1993); Patrash, S. J. and E. T. Zellers, Anal.
Chem. 65:2055-2066 (1993); Rose-Pehrrson et el., Anal. Chem.
60:2801-2811 (1988); Carey et el., Anal, Chem, 59:1529-1534 (1987);
Zellers et. el., Sens. Actuators 12:123-133 (1993); Grate et el.,
Anal. Chem. 60:869-875 (1988); and Grate et al., Anal. Chem.
64:610-624 ( 1992).
In comparison, piezoelectric chemical sensors are quartz crystal
electrodes coated with a polymeric film. The use of such
piezoelectric sensors to investigate fragrances and the nature of
human reactions to different odors is exemplified by Yokoyama, K.
and F. Ebisawa, Anal. Chem, 65:673-677 (1993).
Insofar as is presently known to date, therefore, while the concept
of pattern recognition as an approach for detection of analytes has
been explored as a potential alternative to traditional chemical
sensors and chemical sensing systems which require one sensor for
each analyte or ligand to be detected, all of these prior
investigations have been electrically based and rely upon changes
in electrical signals as the means for detection and evaluation. In
particular, no optical sensor or optical sensing system has ever
been envisioned or constructed which would operate to detect
multiple spectral responses or evaluate them as spectral
recognition patterns. Instead, the conventional guiding principle
and requisite axiom of one specifically binding sensor for each
analyte or ligand to be detected remains rigidly in force, as
demonstrated by the most recent innovations in optical sensors and
optical detection systems conventionally. Accordingly, were an
optical sensor and detection system developed which would be only
semi-selective in its binding and reaction characteristics such
that a single dye reagent would provide a variety of different
spectral responses for multiple analytes and ligands in a manner
which was both accurate and reproducible, such a novel optical
innovation and detection system would be recognized as a major
pioneering advance and achievement over conventional detection
instruments and methods.
SUMMARY OF THE INVENTION
The present invention has multiple aspects, each of which is
substantially related to the others. A first aspect of the
invention provides an optical sensor for detecting at least one
analyte of interest in a fluid sample, said optical sensor
comprising:
a supporting substrate; and
an array formed of multiple semi-selective sensing receptor units
which differ in their constituent chemical formulations, and are
immobilized on said supporting substrate for reactive contact with
the fluid sample, and react semi-selectively with an analyte of
interest, each of said sensing receptor units of said array being
comprised of
a polymeric substance of conventional chemical composition, and
a dye compound of conventional chemical composition which has
characteristic spectral properties, and is disposed in admixture
with said polymeric substance.
(a) wherein said admixed dye compound absorbs light energy of a
predeterminable wavelength and, in the presence of said polymeric
substance without an analyte, yields a baseline spectral response
which is optically detectable and recognizable as showing an
absence of analyte, and
(b) wherein said admixed dye compound absorbs light energy of a
predeterminable wavelength and, in the presence of said polymeric
substance and at least one analyte of interest, generates a
modified spectral response which is optically detectable and
recognizable as showing the consequence of reaction with an analyte
of interest,
said sensing receptor units of said array presenting individual and
alternative modified spectral responses after semi-selective
reaction with an analyte of interest, the overall spectral pattern
formed collectively by said alternative modified spectral responses
for an analyte of interest resulting in spectral recognition
pattern means by which to detect and identify an analyte of
interest.
A second aspect of the invention provides an optical sensing
apparatus for detecting at least one analyte of interest in a fluid
sample, said optical sensing apparatus comprising:
a supporting substrate; and
an array formed of multiple semi-selective sensing receptor units
which differ in their constituent chemical formulations, and are
immobilized on said supporting substrate for reactive contact with
the fluid sample, and react semi-selectively with an analyte of
interest, each of said sensing receptor units of said array being
comprised of
a polymeric substance of conventional chemical composition, and
a dye compound of conventional chemical composition which has
characteristic spectral properties, and is disposed in admixture
with said polymeric substance.
(a) wherein said admixed dye compound absorbs light energy of a
predeterminable wavelength and, in the presence of said polymeric
substance without an analyte, yields a baseline spectral response
which is optically detectable and recognizable as showing and
absence of analyte, and
(b) wherein said admixed dye compound absorbs light energy of a
predeterminable wavelength and, in the presence of said polymeric
substance and at least one analyte of interest, generates a
modified spectral response which is optically detectable and
recognizable as showing the consequence of reaction with an analyte
to interest,
said heterogeneous sensing receptor units of said array presenting
individual and alternative modified spectral responses after
semi-selective reaction with an analyte of interest, the overall
spectral pattern formed collectively by said alternative modified
spectral responses for an analyte of interest resulting in spectral
recognition pattern means by which to detect and identify an
analyte of interest;
means for introducing a fluid sample to said optical sensor for
reactive contact;
means for introducing light energy of a predeterminable wavelength
to said semi-selective sensing receptor units; and
computerized optical detection and evaluation means for optically
detecting each alternative modified spectral response generated by
said heterogeneous sensing receptor units individually and for
evaluating said resulting spectral recognition pattern to determine
the presence of an analyte of interest in the fluid sample.
A third aspect of the invention provides an optical method for
detecting at least one analyte of interest in a fluid sample, said
optical method comprising the steps of:
providing an optical sensor comprised of
a supporting substrate; and
an array formed of multiple semi-selective sensing receptor units
which differ in their constituent chemical formulations, and are
immobilized on said supporting substrate for reactive contact with
the fluid sample, and react semi-selectively with an analyte of
interest, each of said sensing receptor units of said array being
comprised of
(a) a polymeric substance of conventional chemical composition,
and
(b) a dye compound of conventional chemical composition which has
characteristic spectral properties, and is disposed in admixture
with said polymeric substance,
(i) wherein said admixed dye compound absorbs light energy of a
predeterminable wavelength and, in the presence of said polymeric
substance without an analyte, yields a baseline spectral response
which is optically detectable and recognizable as showing an
absence of analyte, and
(ii) wherein said admixed dye compound absorbs light energy of a
predeterminable wavelength and, in the presence of said polymeric
substance and at least one analyte of interest, generates a
modified spectral response which is optically detectable and
recognizable as showing the consequence of reaction with an analyte
of interest,
said heterogeneous sensing receptor units of said array presenting
individual and alternative modified spectral responses after
semi-selective reaction with an analyte of interest, the overall
spectral pattern formed collectively by said alternative modified
spectral responses for an analyte of interest resulting in spectral
recognition pattern means by which to detect and identify an
analyte of interest;
introducing the fluid sample to said optical sensor for reactive
contact;
introducing light energy of a predeterminable wavelength to said
semi-selective sensing receptor units of said optical sensor;
optically detecting each alternative modified spectral response
generated from said sensing receptor units of said optical sensor
collectively to form a resulting spectral recognition pattern;
and
evaluating the resulting spectral recognition pattern using
computerized means to determine the presence of an analyte of
interest in the fluid sample.
BRIEF DESCRIPTION OF THE DRAWING
The present invention may be more easily and completely understood
when taken in conjunction with the accompanying drawing, in
which:
FIG. 1 is a simplified embodiment of the optic sensor comprising
part of the present invention;
FIG. 2 is an illustration of the optic sensing apparatus and
instrumentation of the present invention;
FIG. 3A-3D are graphs illustrating the individual baseline spectral
response in air alone generated by the four different thin film,
sensing receptor units of FIG. 1;
FIG. 4 is a graph showing all the baseline spectral responses of
FIGS. 3A-3D collectively as a unified spectral recognition
pattern;
FIGS. 5A-5D are graphs illustrating the first modified spectral
response generated by the four different thin film, sensing
receptor units of FIG. 1 after reaction with amyl acetate;
FIG. 6 is a graph showing all the first modified spectral responses
of FIGS. 5A-5D collectively as a unified spectral recognition
pattern;
FIGS. 7A-7D are graphs illustrating the second modified spectral
response generated by the four thin film sensing receptor units of
FIG. 1 after reaction with benzene;
FIG. 8 is a graph showing all the second modified spectral
responses of FIGS. 7A-7D collectively as a unified spectral
recognition pattern;
FIG. 9 is an illustration of the instrumentation used
experimentally to test gaseous chemical compositions using the
present invention;
FIG. 10A and 10B are graphs showing alternative modified spectral
responses to different chemical compounds generated by a first
formulated sensing receptor unit;
FIGS. 11A and 11B are graphs showing alternative modified spectral
responses to different chemical compounds generated by a second
formulated sensing receptor unit;
FIGS. 12A and 12B are graphs showing alternative modified spectral
responses to different chemical compounds generated by a third
formulated sensing receptor unit;
FIG. 13 is a graph showing alternative modified spectral responses
to different chemical compounds generated by a fourth formulated
sensing receptor unit;
FIGS. 14A and 14B are graphs showing the effects of different
sequences of chemical reaction upon the alternative modified
spectral responses generated by one sensing receptor unit;
FIGS. 15A and 15B are graphs showing the effects of different
sequences of chemical reaction upon the alternative modified
spectral responses generated another sensing receptor unit;
FIGS. 16A and 16B are graphs illustrating a spectral recognition
pattern indicative for air;
FIGS. 17A and 17B are graphs illustrating a spectral recognition
pattern indicative for amyl acetate;
FIGS. 18A and 18B are graphs illustrating a spectral recognition
pattern indicative for benzene;
FIGS. 19A and 19B are graphs illustrating a spectral recognition
pattern indicative for carbon tetrachloride;
FIGS. 20A and 20B are graphs illustrating a spectral recognition
pattern indicative for ethyl dichloride; and
FIGS. 21A and 21B are graphs illustrating a spectral recognition
pattern indicative for toluene;
FIG. 22 is an overhead view of a prepared optical fiber sensor,
comprising 19 semi-selective sensing receptor units in the
array;
FIG. 23 is an illustration of the instrumentation used
experimentally to test vapor samples using the optical fiber sensor
array of FIG. 22;
FIG. 24 is a graph illustrating a spectral recognition pattern
indicative for benzene using a first grouping of 6 spectral
responses from among the 18 spectral responses generated by the
optical fiber sensor of FIG. 22;
FIG. 25 is a graph illustrating a spectral recognition pattern
indicative for benzene using a second grouping of 6 spectral
responses from among the 18 spectral responses generated by the
optical fiber of FIG. 22; and
FIG. 26 is a graph illustrating a spectral recognition pattern
indicative for benzene using a third grouping of 6 spectral
responses from among the 18 spectral responses generated by the
optical fiber sensor of FIG. 22.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is an optical sensor, apparatus, and
methodology capable of detecting and identifying one or more
analytes or ligands of interest, individually and in mixture, on
the basis of spectral response patterns generated by an array of
thin film, semi-selective, chemical sensing receptor units. Each
formulation of sensing receptor unit comprising the array of the
optical sensor reacts with a plurality of different chemical
compounds and compositions; and for each individual chemical
compound, provides a spectral response pattern over time (by
changes in energy intensity, or by changes in wavelength or both of
these parameters) which is indicative of the event and consequence
of the reaction with a single compound. The array also generates
spectral responses and patterns from mixtures of different
compounds based upon the optical responses from each of the
individual compounds forming this mixture. In this manner, the
optical sensor of the present invention mimics the mammalian
olfactory system in which multiple receptor neurons react with and
respond to the presence of odors by the generation of multiple
neural responses concurrently.
The present invention provides an array of thin film,
semi-selective sensing receptor units which collectively present
multiple spectral responses as a grouped pattern of response
progressions which are monitored over time and are evaluated
collectively as an assemblage of different spectral responses
generated concurrently. The formed collective pattern is then used
as the basis for recognition and identification of an analyte or
mixture of different analytes.
The present invention therefore represents a radical departure from
the conventional approach of optical sensors and detection systems
which demand one sensor for each analyte to be detected; and is a
singular construction and usage of dye reagents and polymeric
materials--each of which is well known, chemically characterized,
and commonly available in the laboratory or from commercial
sources. As such, the present invention pioneers an entirely unique
development both in principle and in construction for the
manufacture and usage of optical sensors and optical detection
systems; and provides the user with major benefits and unforeseen
advantages not previously available heretofore. These include the
following.
1. Specific identification of chemical compounds: The present
invention can identify specific analytes and mixtures of different
ligands by reference to and comparison with previously established
spectral recognition patterns for a variety of known chemical
compounds and compositions. Conventional chemical detection systems
typically discriminate only between different classes of chemical
compounds at best (such as organophosphorous and organosulfur
classes, polynuclear aromatic compounds, and the like). In
comparison, the present invention can identify an analyte of
interest as being a specific chemical compound (such as benzene)
and has the capacity to be constructed such that the sensors and
detection systems of the present invention are capable of
discriminating between homologous compounds or ligands (such as
detecting and distinguishing between benzene and toluene alone or
in admixture).
2. Speed: The optical sensor and detection system uses both
temporal and spectral recognition patterns which require only a few
seconds of time for a complete response progression to be
generated; and require only milliseconds of reaction contact time
as the minimal time intervals for detection and identification
purposes. As noted and described hereinafter, a complete series of
concurrently generated temporal or spectral response progressions
can be obtained in twenty seconds or less which collectively form a
complete spectral recognition pattern for identification purposes.
In comparison, conventionally known sensor systems (including SAW
sensors and piezoelectric sensors), typically require several
minutes (and often 15 minutes or more ), of reaction time in order
to accumulate sufficient empirical data to make a
determination.
3. Reproducibility of results: The optical sensor and detection
systems of the present invention utilize and rely upon recognition
and identification of established spectral responses collectively
as a pattern which is formed from the signals generated by each
thin film sensing receptor unit in the army. Each distinct analyte
or ligand of interest (alone or in admixture) coming in contact
with the array of multiple sensing receptor units will generate a
series of individual spectral responses, repeatedly at each
occasion of contact, so long as the chemical constituents of each
receptor unit remain unaltered. In this manner, each discrete
sensing receptor unit, being a thin film of specified chemical
composition and formulation, yields and provides similar spectral
responses over time at each occasion repeatedly when that
individual analyte or ligand is encountered. Thus, each spectral
response progression from identically formulated sensing receptor
units will be substantially the same after each encounter with the
particular analyte or ligand; and the resulting spectral responses
from the different sensing receptor units will collectively form a
recognizable group or fingerprint pattern by which to detect and
identify that analyte or mixture of ligands routinely and
repeatedly.
4. A library reference paradigm: The optical sensing apparatus and
methodology relies upon and utilizes a prepared library of
reference spectral recognition patterns as the means for evaluating
and identifying the analyte or a mixture of ligands in the fluid
sample. The reference library of spectral recognition patterns is
prepared in advance either by the intended user of the detection
system and/or by the manufacture of the instrumentation using a
diverse variety of different known chemical compounds. Each of the
known chemical compounds or compositions, after reacting with the
multiple semi-selective sensing receptor units comprising the
array, will generate a plurality of spectral responses over
time-which collectively form a spectral pattern of response which
is stored in the memory unit of the apparatus as an established
pattern unique to that compound and is used for recognition
purposes subsequently by the system. The library reference of
established collective patterns may therefore be expanded and/or
customized to meet the demands or requirements of the user or the
specific application circumstances. By intentionally reacting known
compounds with the optical sensor and detection system, sets of
established spectral response progressions and plotted spectral
patterns will be generated which will be compiled and stored as a
reference set of patterns by the system. The reference spectral
recognition patterns will typically be stored in the memory unit of
the instrument; and may be maintained and evaluated mathematically
as a set of algorithms unique for that specific analyte or ligand.
In this manner, any liquid or gaseous sample believed to contain
the analyte or ligand (either alone or in admixture with other
compounds) will provide spectral responses and a spectral
recognition pattern which can then be compared to the previously
established reference patterns and evaluated on this basis to
determine the presence or absence of that specific analyte or
ligand in the sample.
5. Customization to the needs and use circumstances: The present
invention intends and envisions that the optic sensor will provide
an array of thin film sensing receptor units whose formulated
combinations of dye reagent and polymeric material may be varied to
meet a particular application or usage; and optimized for detecting
a singular group or specific class of chemical compounds expected
to be encountered or released in the intended circumstances.
Accordingly, a variety of known compositions and compounds which
are deemed unique for or indicative of a physical circumstance or
location, a hazardous or toxic problem, a clinical or environmental
need, or a singular development setting will be exposed in advance
to the detection system for reaction with the array of multiple
sensing receptor units of the optical sensor to establish a
reference library. In this manner, optical sensors and detection
systems can be manufactured to detect and identify specific
chemical entities which are envisioned or expected to be present in
the test sample. Thus, a single optical system can be used in many
different applications such as: to detect pollutants in air, water,
and soil; to provide qualitative and quantitative measurement of
gases such as carbon dioxide, and oxygen in the blood; to identify
polynuclear aromatic compounds released from combustion engines;
and to monitor specific compounds or elements present during
in-flow processing systems for quality control purposes.
In order to understand and appreciate the different aspects of the
present invention more fully, the invention will be disclosed in
detail by a series of descriptive sections presented seriatim.
These include the array of thin film, semi-selective sensing
receptor units comprising the optic sensor; the apparatus and
instrumentation for optically identifying and evaluating spectral
responses; the range and variety of chemical constituents forming
the thin films of each sensing receptor unit; the functions of the
polymeric substance and the dye reagent forming each thin film
sensing receptor unit; the methodology by which optical detection
and evaluation is made; and a series of experiments and empirical
data which demonstrate the operability and value of the system as a
whole.
I. The Array of Thin Film Semi-Selective Sensing Receptor
Units.
The optic sensor for detecting an analyte or ligand of interest (or
a mixture of these) in a liquid or gaseous sample comprises two
component parts: a supporting substrate; and an array formed of
thin film, semi-selective sensing receptor units which are
heterogenous in that they differ in their constituent chemical
formulations and which are all immobilized on the supporting
substrate for reactive contact with the fluid sample. A highly
simplified construction and exaggerated view of an optic sensor is
shown for clarity of understanding and description by FIG. 1.
The sensor 10 appears in a greatly magnified, highly simplistic
format in order that the basic structural component parts and the
manner of their interaction may be easily grasped. A substantially
rectangular supporting substrate 12 is shown having a substantially
planar or flat external surface 14. Disposed upon the external
surface 14 are a plurality of thin film sensing receptor units 20,
30, 40, and 50 respectively. More than one sensing receptor unit of
the same type (chemical formulation) may be employed; and each type
of sensing receptor unit is formulated differently as a thin film
and has its own unique combination of a polymeric substance and a
dye compound which has well characterized spectral properties. In
each receptor unit, the dye compound has been intermixed with the
polymeric substance as a thin film; and, in the absence of any
analyte or mixture of ligands, is able to absorb light energy of at
least one wavelength and will yield a spectral response over time
(a progression) which is optically detectable and recognizable.
As shown by FIG. 1, the thin films for each sensing receptor unit
20, 30, 40, and 50 have been intentionally and somewhat
artificially drawn to show differences in chemical formulation and
constituents by geometrically different configurations. It is for
this reason and this reason alone that sensing receptor units 20a
and 20b are shaped as circles; sensing receptor units 30a and 30b
are configured as hexagons; sensing receptor units 40a and 40b are
trapezoids; and sensing receptor units 50a and 50b appear as
triangles. In manufacturing tangible embodiments of these different
sensing receptor units, however, it will be appreciated and
understood that there is no restriction or limitation whatsoever
regarding the true configuration, overall surface size, or actual
placement of the different sensing receptor units on the supporting
substrate. The artificial configurations for the different sensing
receptor units shown in FIG. 1 are thus merely illustrative and for
the benefit of the reader in order to be distinguish among the
different sensing receptor units themselves.
Also merely for illustrative and descriptive purposes herein, the
simplest format of differing formulations in dye compound and
polymeric substance have been employed. Accordingly, a single dye
reagent which is a fluorophore (such as Nile Red) has been combined
with four different polymers to form four distinct formulation
combinations. Thus sensing receptor units 20a and 20b are
identified and employ Nile Red dye with the same first polymeric
composition; while sensing receptor units 30a and 30b are identical
and employ Nile Red dye with the same second polymeric substance;
and sensing receptor units 40a and 40b are identical and utilize
the Nile Red dye with the same third polymer; while sensing
receptor units 50a and 50b are identical and employ the Nile Red
dye reagent with the same fourth polymer. Each combination of Nile
Red dye and a different polymeric material forms a unique
combination which is present in duplicate units and is chemically
different and distinguishable from the other formulated
combinations of the adjacent sensing receptor units.
It will be understood and noted that the differing chemical
formulations for each of the sensing receptor units 20, 30, 40, and
50 respectively may be formulated quite differently and distinctly
such that only one dye compound and only one polymeric substance is
used without repetition in any other receptor unit embodiment or
its chemical combination. The range, variety, and diversity of dye
reagents and polymers available and suitable for use in forming
each of the sensing receptor units individually will be described
in detail hereinafter.
The supporting substrate 12, although substantially rectangular in
shape within FIG. 1, may take any size, configuration, or
appearance; may be geometrically regular or irregular; and may be
composed of one or any variety of different materials as the use
occasion requires or permits. As shown within FIG. 1, the
supporting substrate 12 is a translucent or transparent article
such that light energy may pass through without being substantially
altered or hindered. The supporting substrate 12 thus serves at
least as a physical location and a fixed placement for each of the
sensing receptor units 20, 30, 40, and 50. This minimum function of
the supporting substrate may be achieved in one of two formats: the
individual sensing receptor unit may be manufactured and fabricated
as a complete thin film entity; and only as a completely formed
thin film then be positioned and immobilized onto the external
surface 14 of the supporting substrate 12 using a suitable
adhesive, sonic welding, or other means of attachment.
Alternatively, each of the multitude sensing receptor units may be
individually cast and formed in-situ directly on the surface 14
using conventional polymer processing techniques. In such an
procedure, the dye compound and the various monomers or copolymers
are combined in admixture; and this reaction admixture is
polymerized in place directly on the external surface 14 to form
the thin film of the sensing receptor unit as a distinct entity.
The details of such manufacture and the alternatives of adhesion or
in-situ manufacture are described in detail hereinafter.
In addition to using transparent or translucent substrates for
fixing and immobilizing the sensing receptor units, it is also
possible to employ optical fibers as the supporting substrate. In
this case, an optical fiber is coated with a polymer dye
combination on the distal tip. Light can be introduced through the
fiber and the optical signal generated by interaction of light with
the polymer dye combination can return either back through the same
fiber and delivered to a detector or the light signal can go
directly to a detector after passing through the polymer dye
matrix. In one configuration each polymer dye combination is placed
on a separate optical fiber. The individual fibers can then be
bundled into an array of optical fibers and held mechanically so
that the coated tips are presented with the analyte of interest and
the detection scheme can be used to simultaneously or concurrently
monitor all optical fibers. In an alternative manifestation the
individual polymer dye combinations can be disposed on the tip of
an imaging fiber array. Spatial resolution of the imaging array
enables each sensing region to be monitored separately.
II. The Optical Sensing Apparatus and Instrumentation System.
In order to be effectively employed, the optical sensor illustrated
by FIG. 1 is employed with optical apparatus and instrumentation
and utilized as a system in order to detect and identify specific
analytes or ligands of interest. A generalized and representative
optical apparatus and instrumentation system is conventionally
available and preferably employed illustrated by FIG. 2.
Sensor measurements may be performed using the apparatus shown
schematically in FIG. 2 in the following manner: White light from
an excitation source 100 (such as an arc lamp) is collimated;
focused by a lens 101; passed through a nm excitation filter 102;
and focused on an optic sensor 10 via a 10X microscope objective
104. The optic sensor 10 is held in an xyz-micropositioner 106
which allows for fine focusing. Excitation light is transmitted and
illuminates each thin film sensing receptor unit in the array of
the sensor which individually fluoresces in proportion to analyte
concentration. The returning fluorescence light is reflected
90.degree. by the dichronic filter 103; desirably, but optimally
passed through a beam splitter cube 108; filtered at an appropriate
emission wavelength by emission filter wheel 110; and then is
detected by the CCD camera 120. Radiometric measurements are
obtained by monitoring fluorescence while switching between two
excitation filters 102 using the emission filter wheel 110. The CCD
camera typically contains a photosensitive element and is coupled
to an electronic intensifier; which in turn is connected to a
computer having a Video Frame Grabber graphic card that digitizes
and processes the video image. Visual imaging is achieved by using
a CCD video camera to collect the light which is reflected
90.degree. by the beam splitter cube. Illumination for visual
imaging purposes is achieved either by rotating the excitation
filter wheel to an empty position (using neutral density filters as
necessary); or by illuminating the sample and its environs at the
distal end of the sensor with an independent light source.
The optic sensing apparatus and instrumentation system shown by
FIG. 2 detects changing fluorescence either as changes in light
intensity or changes in light wavelength over time-that is, a
spectral response progression generated by and released from each
individual sensing receptor unit after initial illumination with
light energy of a pre-determined wavelength which is then absorbed
by the dye compound in each thin film receptor unit. The light
energy emitted from each sensing receptor unit individually (in the
presence of and in the absence of a ligand or analyte of interest),
is collected using a CCD video camera using standard frame grabbing
technology and image processing capabilities. Each spectral
response progression detected as emission light energy by the
detector of the CCD is recorded; and the pattern of fluorescence is
shown either as changes in energy wavelength or as different light
intensity pixels on the detector representing the spatial
dimension. By definition, a pixel is a picture element--a sensitive
region--which determines light intensity and/or light energy
quantum. In the experiments described and presented hereinafter,
each dye compound/polymeric substance combination comprising the
thin film of each sensing receptor unit was placed under the
objective lens of the epi-illuminating fluorescence microscope; and
observed sequences of 33 and 64 milliseconds duration received as
video images obtained at 300 millisecond intervals were acquired
and placed in the memory of the computer. In this manner, each
video sequence showing the light intensity response progressions
from each sensing receptor unit lasted about 19 seconds in
total.
III. The Spectral Response Progressions and the Collective Response
Patterns.
The optic sensor 10, in combination with the apparatus and
instrumentation system of FIG. 2, presents an array where each of
the sensing receptor units generates a spectral response over time
which is detectable and identifiable as a progression of changes in
emitted light energy intensity or in emitted light wavelength.
Thus, the admixed dye compound of the thin film absorbs exciting
light energy of a predeterminable wavelength and, in the presence
of the polymeric substance and without any analyte or ligand,
yields a baseline spectral response progression over time which is
optically detectable and recognizable as showing an absence of
analyte. This characteristic result shown as changes in light
intensity is illustrated by FIG. 3.
It will be recalled that there are a pair of identical sensing
receptor units 20a and 20b, each of which is a thin film of the
same dye compound and polymeric substance in admixture. Each
receptor unit 20 will therefore absorb exciting light in the
presence of air in the ambient environment; at a substantially
constant intensity and then emit fluorescent light as a spectral
response. The emitted light energy will not meaningfully fluctuate
and change in intensity over time in the presence of air alone
(without the presence of any specific analyte or ligand). This is
illustrated by FIG. 3A which shows a spectral response progression
over approximately 10 seconds duration as an effectively flat or
substantially constant plot in which the fluorescence does not
change in light intensity to any meaningful degree. The dotted
curve of FIG. 3A shows the identical patterns generated by each of
the receptor units 20a and 20b cumulatively.
In comparison, sensing receptor units 30a and 30b are identical
thin films of a differently formulated combination of dye compound
and polymeric substance. When exciting light is introduced to
receptor units 30 in air alone, the emissions detected as
fluorescent light also do not fluctuate or change over time as
illustrated by FIG. 3B. As noted, the plot of spectral response
does not meaningfully change over the 10 second duration. FIG. 3b
represents the progressions of individual responses generated from
sensing receptor units 30a and 30b individually. It is noted and
appreciated, however, that the spectral response progression over
time generated by receptor units 30 and shown by FIG. 3B is
different and distinguishable from that spectral response
progression generated by sensing receptor units 20 illustrated by
FIG. 3A.
A similar result is revealed as regards sensing receptor units 40
and sensing receptor units 50a and 50b respectively. Sensing
receptor units 40 also differ in their specific formulation of dye
compound and polymeric substance forming the thin film of the unit.
When illuminated by exciting light energy of a predeterminable
wavelength in the presence of air alone, these units yield light
emissions which are substantially uniform and constant over time.
These are cumulatively shown by FIG. 3C as the spectral response of
light intensity over ten seconds duration. However, it will be
recognized that the spectral response of light intensity of FIG. 3C
does not vary greatly or substantially from either of those shown
by FIGS. 3A and 3B respectively.
Finally, the spectral response in air alone of the (differently
formulated) sensing receptor units 50a and 50b are illustrated by
FIG. 3D. This dye compound and polymeric substance formulation
differs from those in the other units in the array; and, in the
presence of air alone, the introduction of exciting light
wavelengths absorbable by the thin films of receptor units 50a and
50b yields a spectral response progression of fluorescent light
intensity over time which shows no meaningful changes and
fluctuations. The overall spectral response of FIG. 3D, albeit
individual, is not markedly different or divergent from those
illustrated by FIGS. 3A, 3B, 3C respectively.
The individual spectral response progressions of FIGS. 3A-3D
respectively are graphically plotted collectively as a group in
FIG. 4. Note that although four distinct plots may be discerned in
FIG. 4, all the individual spectral responses generated by the four
types of sensing receptor units are effectively alike and
substantially uniform. The collective total and cumulative plot of
all four spectral response progressions together yield a unique
pattern of spectral responses over time--a fingerprint--which
recognized as the distinctive collective result and effect caused
by the array of heterogeneous sensing receptor units 20, 30, 40,
and 50. FIG. 4 thus illustrates a spectral recognition pattern
which is indicative and recognizable as a baseline pattern
indicative of the presence of any analyte or ligand; and is the
distinctive representational total of all the spectral responses
from each of the thin film sensing receptor units forming the
array. For detection and evaluation purposes, the spectral
recognition pattern illustrated by FIG. 4 is stored in the retained
memory of the computerized instrumentation and system of FIG. 2;
and this initial spectral recognition pattern showing the response
progressions serves as the spectral baseline and means for
evaluation, as well as for recognizing, and identifying the
presence or absence of an analyte or ligand in the testing
system.
A very different response and spectral pattern is produced when a
first analyte of interest is introduced to the array of
semi-selective sensing receptor units of the optical sensor 10.
Under these circumstances, the dye compound in each thin film
receptor unit absorbs light energy of a predeterminable wavelength
and, in the presence of the polymeric substance and the first
analyte of interest, generates a first modified spectral response
showing changes in light intensity over time which is optically
detectable and is the consequence of reaction with the first
analyte of interest. This fluctuation in light intensity phenomenon
and result is illustrated by FIG. 5 which shows the effect of
introducing amyl acetate to each of the sensing receptor units 20,
30, 40, and 50 of the optical sensor 10 illustrated within FIG.
1.
Sensing receptor units 20a and 20b each absorb exciting light
energy in the presence of amyl acetate; and the resultant changes
in fluorescent light intensity are detectable as major fluctuations
over time as shown by FIG. 5A. The spectral response yielded by
receptor units 20a and 20b in the presence of the amyl acetate
analyte as illustrated by FIG. 5A is remarkably different and
distinct from the baseline pattern shown previously by FIG. 3A. The
modified light intensity response progression over time is observed
and evaluated as being markedly different and unique.
A comparable consequence and result occurs for each of the other
sensing receptor units 30, 40, and 50 respectively. A first
modified spectral response to the presence of amyl acetate for
receptor units 30a and 30b is revealed by FIG. 5B--a =response
which fluctuates in light intensity and is very different from that
baseline response shown by FIG. 3B. Similarly, the first modified
spectral response progression over time for receptor units 40a and
40b is illustrated by FIG. 5C; and shows a very different response
to the presence of amyl acetate from the baseline progression
illustrated by FIG. 3C. Similarly, in the presence of amyl acetate
the first modified spectral response over time generated by
receptor units 50a and 50b in light intensity over time is shown by
FIG. 5D as a substantial variation and fluctuating difference from
the baseline spectral response of these sensing receptor units
revealed previously by FIG. 3D.
The collective total of all four modified spectral response
progressions over time from FIGS. 5A-5D together is presented by
FIG. 6 as a plotted spectral pattern which is easily recognized as
different and distinct from the established baseline pattern shown
by FIG. 4. Clearly, the spectral recognition pattern of FIG. 6 as
an overall pattern of four plots or progression curves is easily
discernible; can be placed in the memory of the computerized
instrumentation; and retained as an established recognition pattern
and reference which is identifiable as the spectral recognition
pattern specific for amyl acetate. Thus, at any time in the future,
one may optically detect and identify the presence of amyl acetate
in a liquid or gaseous sample easily and quickly by comparing the
established spectral pattern of FIG. 6 with the baseline pattern
illustrated by FIG. 4. This direct comparison of retained (and
established reference) spectral recognition patterns as a baseline
(shown by FIG. 4) and for amyl acetate (shown by FIG. 6) allows for
accurate, repeated, and speedy detection and identification of amyl
acetate using the optical sensor and that specific set of
semi-selective sensing receptor units.
The optic sensing apparatus and detection instrumentation system
also intends and provides the capability of detecting and
identifying multiple analytes or ligands in admixture when
introduced together in a liquid or gaseous fluid sample. Using the
optical sensor of FIG. 1 and the instrumentation system of FIG. 2,
when a second analyte (such as benzene) is introduced to the
optical sensor, the overall system operates to provide a second and
entirely different result. The admixed dye compound of the thin
film in each of the sensing receptor units individually absorbs
exciting light energy of a predeterminable wavelength; and, in the
presence of the polymeric substance and a second analyte of
interest such as benzene, generates a second modified spectral
response progression (measurable either as changes in light
intensity or as changes in light wavelength over time ) which is
individually optically detectable and individually recognizable as
showing the consequence of reaction with the second analyte of
interest, benzene. This is graphically illustrated by FIG. 7.
As is shown therein, the sensing receptor units 20a and 20b, in the
presence of benzene, absorb exciting light energy and then emit
fluorescent light energy which fluctuates and changes in intensity
over time and provides a second modified spectrum response
progression as shown by FIG. 7A. This second modified spectral
response progression of FIG. 7A, the consequence of benzene
reaction, is remarkably different and readily distinguishable from
that first modified spectral response progression over time for
amyl acetate illustrated by FIG. 5A; and is also markedly different
and separable from the baseline spectral response illustrated by
FIG. 3A. It will be appreciated that in each instance, the same
thin film sensing receptor units 20a and 20b were repeatedly
employed and utilized. Nevertheless, the same sensing receptor
units 20a and 20b yielded three completely different spectral
response progressions over time after interaction with air alone,
after interaction with amyl acetate, and after interaction with
benzene. The semi-selectivity of the sensing receptor units 20a and
20b are thus able to react differently, semi-selectively, and
demonstrate a different photokinetic result for each of the three
chemical compositions. The sensing receptor units are thus able to
generate entirely different spectral responses as a consequence of
reaction with each of the three chemical entities.
A similar consequence and result occurs for each of the sensing
receptor units 30, 40, and 50 respectively. The effect of reaction
with benzene by the thin films of receptor units 30a and 30b and
the resulting change in light intensity (or light wavelength), the
spectral response over time is shown by FIG. 7B--which is markedly
different from the first modified spectral response progression
yielded for amyl acetate illustrated by FIG. 5B, and is also
distinguishable from the baseline spectral response shown by FIG.
3B. The same overall result and consequence of reaction with
benzene by receptor units 40a and 40b is illustrated by FIG. 7C;
and the consequences of benzene reaction with receptor units 50a
and 50b is illustrated by FIG. 7D. In each instance, each of the
sensing receptor units show a second modified response over time
after reaction with benzene in comparison to the reaction with amyl
acetate or the presence of air alone.
The collective sum of all four individual second modified spectral
response progressions over time yielded by the entire array of
sensing receptor units is illustrated by FIG. 8 as a distinctive
group or assemblage pattern. It will be appreciated that the
overall pattern formed by all the optical responses plotted over
time in the presence of benzene shown by FIG. 8 is easily
recognized and quickly distinguishable from that collective group
pattern generated for amyl acetate shown by FIG. 6; and is also
easily separable and distinguishable from the collective baseline
pattern for air alone illustrated by FIG. 4. The group pattern of
FIG. 8 generated by the entire array of multiple thin film sensing
receptor units collectively is then preferably entered and retained
in the memory of the computerized instrumentation and thus becomes
part of the established reference library for the instrument and
system. In this manner, the library now has retained spectral
recognition patterns for three distinct chemical substances (air,
amyl acetate, and benzene) using the same optical sensor and
instrumentation throughout. Therefore, any fluid sample to be
tested subsequently comprising air, or amyl acetate, or benzene
alone can be optically detectable and identifiable as individual
chemical compounds routinely and reproducibly. Similarly, a fluid
sample which comprises a mixture of air, amyl acetate and benzene
in combination can also be detected and recognized because the
response will contain components of each spectral recognition
pattern shown by FIGS. 4, 6, and 8 respectively.
FIGS. 3-8 and the specific entities of air, amyl acetate and
benzene respectively will be understood to be merely illustrative
examples showing how the essential parts of the system function and
representationally disclosing how the multiple spectral responses
generated by the array of multiple sensing receptor units are
generally employed to form collective group patterns of spectral
responses for recognition and identification purposes. Accordingly,
these entities embody and demonstrate the underlying principles for
practicing the present invention as a whole; and provide only the
most basic and fundamental statement and description of the
intended range of present invention common to the formats,
different modes of operation, and envisioned diversity of
applications for the system.
IV. The General Operating and Controlling Parameters of the
Invention.
In order to be both effective and efficient as a methodology and
instrumentation system able to detect and evaluate one or more
analytes or ligands of interest, there are a number of operating
parameters and controls which should be followed and incorporated
into the system in order to obtain useful or optional results.
These include the following:
1. Detectable Entities and Species:
The present invention has the capability to be used in alternative
detection modes. For example, conventionally known individual
analytes or ligands can be detected and identified as single
chemical compounds when present in a liquid or gaseous sample.
Alternatively, blends or admixtures of different analytes or
ligands in one .fluid sample can also be identified as a mixture of
distinct formulated entities or chemical species. In addition, the
component compounds of entirely novel chemical compositions and
formulations never previously synthesized and/or never
characterized may be analyzed to yield detectable features on
chemical moiety information using the components of the present
invention. This range of system capabilities allows the user to
obtain useful chemical information not only about the specific
entities and species, but also permit analysis and determination of
chemical component parts and moieties of compositions which are
themselves entirely novel and unique.
2. The Nature of the Spectral Response:
The present invention intends and permits the user a choice of
spectral properties to be employed as the feature for making
determination and identifications. The user may, at his option,
utilize either light energy intensity or light energy wavelength as
the parameter to be detected and measured. Thus, a spectral
response over time from a thin film sensing receptor unit can be
received as the changes in light intensity over a set time
duration; and, alternatively, the spetral response over time to be
detected and measured can be the changes in light wavelength over
time. If the user so desires, on a very critical analysis if
necessary, both forms of spectral response can be performed
concurrently, simultaneously, or sequentially.
3. A Preferred Instrumentation.
A preferred apparatus and instrumentation of the optical detection
system will utilize a computerized central processing unit and
computerized controls for performing the different functions of
directing exciting light energy to the correct location; detecting
the changes in light intensity and/or light energy wavelengths; and
plotting a spectral response progression from each of the thin film
sensing receptor units individually. These tasks can be performed
concurrently, if not simultaneously, by a computerized network
system which not only will collect the raw empirical data as a
series of fluctuating fluorescent light emissions but also record
and remember them individually in order that the data may appear as
recognizable spectral patterns retained for identification and
evaluation and stored internally within the reference library set
of recognition patterns. The ability to detect, record, and
remember the individual spectral response progressions as well as
the ability to generate collective patterns and the ability to
employ and compare sets of spectral recognition patterns
consecutively in series in order to detect and identify specific
chemical compounds is best performed in a minimum of time and with
maximal efficacy using computerized systems.
4. The Establishment of a Reference Library.
The preferred optical sensing apparatus and instrumentation system
requires the development and the presence of a reference library of
established spectral recognition patterns for each analyte or
ligand which is to be detected by the system. Since the optic
sensor provides an array of multiple, semi-selective sensing
receptor units--which can be altered markedly in the combinations
of dye compound and polymeric substance in admixture-the reference
library set of established patterns should reflect the prior test
experience and recorded results of using that array of sensing
receptor units of specified chemical formulation, Clearly, by
altering the chemical constituents (the particular combination of
dye reagent and/or polymeric substance in any one or more of the
sensing receptor units), the spectral responses over time will be
changed; and the previously established spectral recognition
patterns associated for known ligands with one established array of
sensing receptor units cannot be meaningfully transferred to or be
used by another set of differently formulated sensing receptor
units. Accordingly, for each array, an established individual
reference library desirably should be established which is personal
to and unique for the thin film units of that specific optical
sensor. Any change in the chemical formulation of any sensing
receptor unit will therefore necessitate a change and alteration in
the reference library set of established spectral recognition
patterns by which identification is made.
5. Detection of Conventionally Known Chemical Entities.
For detecting and identifying well known chemical compounds and
species suspected of being present in a liquid or gaseous fluid
sample using the present optic sensing apparatus and
instrumentation system, the total number of distinct analytes or
ligands (alone or in admixture) which can be detected and
identified as specific chemical compounds or discrete compositions
is limited; and requires that the specific chemical substance have
been tested previously and have an established spectral recognition
pattern in the reference library set for that particular sensor.
Thus, if a fluid sample is introduced to the testing system in
which ten different and unrelated chemical compounds are intermixed
and, only 5 of these have established spectral recognition patterns
within the reference library of the instrument, that system
typically will only be able to detect only those 5 particular
chemical compounds out of the 10 entities actually present in the
sample. Accordingly, it will be recognized that the total number of
previously established spectral recognition patterns for known
chemical substances is a major factor in that the apparatus; and
the system typically cannot detect or identify a distinct chemical
entity or species as such which it has not previously encountered
and established as a collective pattern spectrally beforehand. The
apparatus and instrumentation thus requires preknowledge and a
prepared spectral background of collective pattern characteristics
before any specific chemical compound or species can be identified.
However, once a series of individual spectral responses over time
and a spectral recognition pattern is established and stored in the
reference library of the instrument, that system will be able to
identify and detect the analyte, or ligand or species of interest
routinely without error.
6. Range and Diversity of Direction.
As a function and consequence of the freedom to alter, control, and
tailor the optic sensor and the array of heterogeneous,
semi-selective sensing receptor units in their individual
formulated combinations of dye compound and polymeric substance to
meet specific use applications and environments, the optical
sensing apparatus and instrumentation system can be varied
radically and be used in a diverse range of different applications.
This range and variety provides the user with the ability to meet
the demand of optically detecting and identifying specific classes
of chemical compounds or chemical species alone or in mixture. It
is therefore expected that various, dedicated optic sensors will be
programmed and used for individual purposes and applications; and
that the optic sensor will be replaced and substituted by another
when the apparatus and instrumentation system is employed for an
alternative or very different usage.
V. Analytes Detectable By An Array Of Heterogeneous Sensing
Receptor Units.
The range and diversity of analytes or ligands which may be
detected and identified, singly or in combination, by the present
invention comprises noxious organic and inorganic compounds in
liquid or gaseous form which are volatile in nature; toxic and
non-toxic gases; environmental pollutants in air, water, and soil;
and any matter which can be dispersed, disaggregated, suspended, or
otherwise carried in a fluid medium. A representative, but
non-exhaustive general listing is given by Table 1 below.
TABLE 1 ______________________________________ Generally Detectable
Analytes (Illustrative Listing)
______________________________________ aromatics thiols benzene
alkyl thiols having from 1-25 carbon atoms toluene alkylbenzenes
halogenated organic carboxylic acids compounds methylene chloride
carboxylic acids, chloroethylene saturated and unsaturated, having
from 1-25 carbon atoms trichloroethylene bromorcetate
trichloromethane fluorohexane chloroform bromoform fluoroform
esters hydrocarbons alkylacetates, alkyl all alkanes, alkenes, and
alkynes propionates, alkyl having from 1-25 carbon atoms butyrates,
etc. metallic salts of fatty acids alcohols and amines all primary,
secondary, and tertiary organic alcohols and amines and homolog
series from 1-25 carbons aldehydes and ketones formaldehyde
acetaldehyde methyl ethyl ketone
______________________________________
The analytes which may be optimally detected and measured using the
present invention individually and collectively often share
characteristics and properties in common. A first and frequently
found property is that the analyte or ligand have a discernible
polarity. Such polarity includes the polarity of bonds caused by
two atoms joined by a covalent bond which share electrons unequally
as well as the polarity of molecules which occurs when the center
of negative charge lies within the molecular structure and thus
constitutes a dipole.
A second commonly shared characteristic of the ligand or analyte is
that they may be in any fluid state--that is in a gaseous, liquid,
or even in a fluid semi-solid state. The majority of analytes or
ligands suitable for detection and identification by the present
invention are expected to be primarily in the vaporous and liquid
physical states--and thus be fluid as a concomitant property of
their existence. However, many organic compounds which typically
exist as solids may also be detected and identified via the use of
solvents and solvent mixtures as these are conventionally known and
used in the scientific literature. Organic solvents and aqueous
solvents are well known and generally employed in research and
industry; and the suspension, dissolution, and/or displacement of a
solid chemical compound by an appropriate solvent will prepare and
present a fluid sample suitable for evaluation and analysis by the
present invention.
A third common property shared among the analytes and ligands is
that they be absorbed and at least partially partitioned by the
polymeric material of the thin film; and that the absorbed and
partitioned analyte or ligand present in the polymeric material
layer meaningfully alter or modify the baseline set of spectral
properties generated by the interaction of a dye with the polymeric
material which exists prior to introduction of the analyte or
ligand of interest. This trait and attribute embodies the
semi-selective reactivity feature of the thin film sensing receptor
units; and provides the different multiple spectral responses which
form the collective recognition patterns themselves.
A fourth feature among the membership of organic analytes and
ligands is that they are predominantly but not exclusively
hydrocarbons. Such compounds are composed primarily of carbon and
hydrogen atoms; but often also contain one or more heteroatoms
selected from the group consisting of nitrogen, oxygen, sulfur,
phosphorous, and halogen atoms. These hydrocarbons, with or without
one or more heteroatoms, may be saturated or unsaturated; may take
shape as linear, branched, ring, or polycyclic structures; and
present formats which include aliphatic, aryl and alkyl aryl
structures or combinations of these. Moreover, it is intended and
expected that the hydrocarbon molecule as a whole, exclusive of any
heteroatoms which may optionally be present, will comprise from 1
to about 25 carbon atoms in total; and that within this range of
carbon atoms, with one or more degrees of unsaturation; as linear,
branched, and ring entities; and in multiple structural formats
will be present.
In general, regardless of the particular molecular weight of the
entity which is to be detected using the present invention, any
analyte or ligand which can penetrate and be captured by the
polymeric material of the thin film sensor (and thus be absorbed
and partitioned during its migration) is suitable for detection
using the present invention. The differences among the various
hydrocarbons and other organic compounds are deemed to be only in
the magnitude of their individual effects upon the dye; and in the
time required for the sensor to respond spectrally to the presence
of the analyte or ligand within the fluid sample.
To demonstrate, a representative but preferred range of
hydrocarbons from petroleum sources suitable for detection by the
present invention are in the listing of Table 2 provided below.
TABLE 2
Hydrocarbons from Petroleum Sources Suitable for Detection
Aromatics such as benzene, toluene, the xylenes, ethyl benzene,
naphthalene, anthracene, phenanthrene, plus their hydrocarbon
derivatives;
Alicyclics (saturated cyclics) such as cyclohexane, tetralin, and
their hydrocarbon derivatives;
Paraffins (branched and straight chain) such as propane; normal and
isobutane; all paraffinic isomers of C5, C6, C7, C8, C9, and
C10;
Olefins such as propylene; the butylenes; all olefinic isomers of
C5, C6, C7, C8, C9, and C10;
Halogenated hydrocarbons comprising chlorine, bromine, fluorine, or
iodine; and Hydrocarbons of up to 25 carbon atoms containing one or
more carbonyl groups (--CO) forming aldehydes and ketones.
VI. The Chemical Constituents of the Thin Film Comprising Each
Sensing Receptor Unit.
Each semi-selective sensing receptor unit forming the heterogeneous
array is a thin film of carefully controlled and limited thickness;
is a structure formed by the polymer and dye reagents including dye
reagents and monomer mixtures, copolymerized dyes, trapped dyes,
and absorbed dyes; and will appear as a discrete film having a
thickness ranging from about 0.01-1,000 microns, a thickness of
between 1-15 microns being most preferred.
Moreover, the thin films themselves may be either transparent or
translucent. Since a thickness of 0.01-1,000 microns is
permissible, none of the thin films demonstrate any substantial
optical density (i.e., absorbance). Thus, highly colored materials
provide thin films which are virtually transparent because of the
short optical path lengths involved.
The polymer comprising a thin film typically is a regular geometric
form, substantially flat and smooth, and can present some polymer
texture or surface irregularity. Alternative physical
configurations, however, are envisioned and intended; such
alternatives would include physical configurations shaped as hairs,
sleeves, wells or cavities, and irregularly shaped embodiments. The
flexibility and tensile strength of the thin film may vary markedly
and often will depend upon the polymer actually used. These
variances are provided and controlled by the user's particular
choice of composition from among the conventionally known different
polymer compositions; and by selective choice of parameters such as
cross-linking concentration, functional group modifications and the
like.
A. The Light Energy Absorbing Dyes Generally Useful:
At least one light energy absorbing dye is disposed in admixture
with a polymeric substance to form the thin film which is the
sensing receptor unit.
Each light energy absorbing dye formulation or composition suitable
for use will react semi-selectively with each analyte or ligand of
interest which is present either alone or in admixture with other
entities. Moreover, each dye will then show evidence of such
semi-selective reactive contact by either absorbing and reflecting
a portion of the light energy; or, alternatively, by absorbing
light energy and then subsequently emitting light energy of a
different wavelength in return. Such reflected or emitted light
energy is conveyed from the thin film; and such conveyed light will
emerge from the array surface for detection and measurement by
intensity or by wavelength of light.
The dyes which may be generally employed and disposed individually
within the thin film are all conventionally known and often
commercially available. The present invention intends that all the
commonly useful properties and capabilities of the various classes
of light energy absorbing dyes be employed as needed or desired for
the specific use or application. Merely illustrative of the many
different dyes are those fluorophores and chromophores listed below
within Tables 3 and 4 respectively.
TABLE 3 ______________________________________ Excitation
Fluorescence Wavelength emission (range or range Compounds maximum)
(max) ______________________________________ A. Fluorophores Eosin
520-530 nm 530-580 nm (550 nm) TRITC-amine 555 nm 570-610 nm (590
nm) Quinine 330-352 nm 382-450 nm Fluorescein W 488-496 nm 530 nm
Acridine yellow 464 nm 500 nm Lissamine Rhodamine 567 nm 580 nm B
Sulfonyl Chloride Erythroscein 504 nm 560 nm Ruthenium 460 nm 580
nm (tris, bipyridium) Texas Red 596 nm 615 nm Sulfonyl Chloride
B-phycoerythrin 545, 565 nm 575 nm (Nicotinamide adenine 340 nm 435
nm dinucleotide (NADN) Flavin adenine 450 nm 530 nm dinucleotide
(FAD) Carboxy 587 nm 640 nm Seminaphthorhodafluor
Naphthofluorescein 594 nm 663 nm
______________________________________
TABLE 4 ______________________________________ Range Chromophores
(max) ______________________________________ Iron-salicylate
complex 530 nm Indamine dye 590 nm INT formazon dye Hopkins-Cole
dye 560 nm Quinone-imine dye 500 nm Fe(SCN).sup.+2 460 nm Malachite
Green 620 nm 4-bromo A-23187, 340 nm Cresol red 415 nm, acid; 570
nm, base diphenylcarbazone 575 nm disulphonic acid Chrome bordeaux
B 575 nm Calmagite 650 nm Ninhydrin dye 650 nm
______________________________________
B. The Preferred Polarity-Sensitive or Solvachromic Dye.
Solvachromic dyes, regardless of specific composition and
formulation are preferred for use; and are identified and defined
in operational terms as a light energy absorbing substance whose
absorption and/or emission spectra are sensitive to and altered by
the polarity of their surrounding environment-including gaseous,
liquid, and/or solid molecules and ions which are temporarily or
permanently present in the immediately adjacent spatial volume. The
term "solvachromic" is derived from the recognized and long
established characteristics of many fluorophores whose fluorescence
emission spectra are sensitive to the polarity of the solvents in
which they are employed or found. For example, if the emission
spectrum of a fluorophore such as ANS
(1-anilino-9-naphthalenesulfonyl acid) is examined in different
solvents of varying polarity, one finds that the emission spectrum
shifts to shorter wavelengths (blue shifts) as the solvent polarity
is decreased. Conversely, increasing solvent polarity generally
results in shifts of the emission spectrum of the fluorophore to
longer wavelengths (red shifts). Red shifts are often, but not
always, accompanied by a decrease in the quantum yield or total of
photons emitted for the fluorophore being evaluated. This
phenomenon, the change in emission spectrum of many fluorophores
with respect to different solvents of varying polarity, is well
described by the following publications: Joseph R. Lakowicz,
Principles of Fluorescence Spectroscopy, Chapter 7, Plenum Press,
New York, 1983, pp. 187-255; Mataga et al., Bull. Chem. Soc. Jpn.
29:465-470 (1956); Bakhishiev, N. G., Opt. Spectrosc. 10:379-384
(1961), and Opt. Spectrosc. 12:309-313 (1962), and Opt. Spectrosc.
13:24-29 (1962); MacGregor, R. B. and G. Weber, Proc. N.Y. Acad.
Sci. 366:140-154 (1981).
While the best known examples of solvachromic dyes are
fluorophores, the membership of this class as a whole includes both
absorbers or chromophores as well as fluorescent molecules. The
essential property common to each and every member of this class of
dyes is that the chosen dye substance changes its spectral
properties when exposed to different solvents of varying polarity.
For fluorophores, this spectral change can include either a change
of emission intensity or a change in the wavelength of the emitted
fluorescent light. For an absorber or chromophore dye, the
intensity of color may shift either toward the red or the blue end
of the spectrum. To determine whether a chosen dye composition is a
member of the class defined as a solvachromic dye, the test is
solely an empirical one. When the dye is exposed to different
organic solvents of varying polarity, the dye changes its color
which is empirically observed as a spectral change. Thus, an
absorber dye demonstrates a spectral change through its color,
either by altering the intensity of the color or by the observation
of an actual color change. Alternatively, a fluorescent dye
demonstrates its sensitivity to different solvents of varying
polarity through changes in either its absorbing exciting light; or
by a change in wavelength of the emitted light; or by a change in
the intensity of the emitted light.
By this operational definition and the empirical test method
through which any person of ordinary skill in this art may identify
a chosen dye substance as being a solvachromic dye, it will be
recognized and appreciated that the terms "solvachromic" and
"polarity-sensitive dye" defines and identifies a dye formulation
which is not only sensitive to different solvents of varying
polarity, but also to any other organic entity, molecule, or
substance which has a discernible,--that is, a demonstrable or
determinable polarity. Thus, organic compositions, compounds, and
formulations of varying polarity which are not solvents as such are
clearly encompassed and included by this term in addition to those
compositions which are classically defined as "organic solvents;"
and organic solvents constitute merely one group or family within
the membership as a whole forming the class of organic analytes
having a discernible polarity. In this manner, while it is most
convenient to test and evaluate a chosen dye using a plurality of
solvents of varying polarity to empirically demonstrate that the
chosen dye is spectrally influenced and altered by the polarity of
the surrounding environment, any other kind or type or organic
molecule may also be employed to demonstrate the spectral
sensitivity of the chosen dye-albeit under less convenient and/or
more rigorous test conditions.
To demonstrate the range and diversity of the membership comparing
the subclass as a whole which constitutes polarity-sensitive or
solvachromic dyes, a non-exhaustive listing of representative
examples is provided hereinafter by Tables 3 and 4 respectively.
Table 5 provides a representative list of polarity-sensitive
fluorophores. Correspondingly, Table 6 provides a range of
illustrative examples which are polarity-sensitive absorber or
chromophoric dyes.
TABLE 5
Polarity-Sensitive Fluorophores.
Phospholipid Fluorophores
N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)
dipalmittcyl-L-a-phosphatidylethanolamine (NBD-PE)
N-(5-fluoresceinthiocarbamoyl)
dipalmitoyl-L-a-phosphatidylethanolamine triethylammonium salt
(TITC DPPE)
N-(Lissamine rhodamine B sulfonyl)
dipalmitoyl-L-a-phosphatidylethanolamine triethylammonium salt
(rhodamine DPPE)
N-(Texas Red sulfonyl) diolsoyl-L-a-phosphatidyle-thanolamine
triethylammonium salt.
N-(Texas Red Sulfonyl) dipalmitoyl-L-a-phosphatidyle-thanolamine
triethylammonium salt (Texas Red DPPE)
3-palmitoyl-2-(1-pyrenedecanoyl)-L-a-phosphatidyl-choline
(10-py-PC)
N-(5-dimethylaminonaphthalene-1-sulfonyl)
dipalmitoyl-L-a-phosphatidylethanolamine triethylammonium salt
N-(1-pyrenesulfonyl) dipalmitoyl-L-a-phosphatidyle-thanolamine
triethylammonium salt
N-(6-(5dimethylaminonaphthalene-1-sulfonyl) amino
)-hexanoyldipalmitoyl-L-a-phosphatidylethanolamine triethylammonium
salt
N-(biotinoyl) dipalmitoyl-L-a-phosphatidylethanolamine
triethylammonium salt
Anionic Fluorophores
cis-parinaric acid
trans-parinaric acid
p-((6-phenyl)-1,3,5-hexatrienyl) benzoic acid (DPH carboxylic
acid)
3-(p-(6-phenyl)-1,3,5-hexatrienyl) phenylpropionic acid (CPH
propionic acid)
1-pyrenecarboxylic acid
1-pyrenebutanoic acid (pyrenebutyric acid)
1-pyreneonanoic acid
1-pyrenedecanoic acid
1-pyrenedodecanoic acid
1-pyrenebexadecanoic acid
11-((1-pyrenesulfonyl) amino) undecanoic acid
2-(9-anthroyloxy) palmitic acid (2-AP)
2-(9-anthroyloxy) stearic acid (2-AS)
3-(9-anthroyloxy) stearic acid (3-AS)
Table 3--Continued
6-(9-anthroyloxy) stearic acid (6-AS)
7-(9-anthroyloxy) stearic acid (7-AS)
9-(9-anthroyloxy) stearic acid (9-AS)
10-(9-anthroyloxy) stearic acid (10-AS)
11-(9-anthroyloxy) undecanoic acid (11-AU)
12-(9-anthroyloxy) stearic acid (12-AS)
12-(9-anthroyloxy) oleic acid (12-AO)
16-(9-anthroyloxy) palmitic acid (16-AP)
9-anthracenepropionic acid
9-anthracenedodecanoic acid
1-perylenedodecanoic acid
6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino) haxanoic acid (NBD
hexanoic acid)
12-(N-methyl)-N-((7 nitrobenz-2-oxa-1,3-diazol-4-yl) amino)
dodecanoic acid
12-(N-methyl-N-((7-nitrobenz-2-oxa-1,3-diazol4-yl) amino)
octadecanoic acid
12-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-dodecanoic
acid
11-(9-carbazole) undecanoic acid (11-CU)
11-((5-dimethylam inonaphthalene-1-sulfonyl)amino)un-decanoic
acid
5-(N-dodecanoyl) aminofiuorescein
5-(N-hexadecanoyl) aminofiuorescein
5-(N-octadecanoyl) aminofiuorescein
5-(N-hexadecanoyl) aminoeosin
1-anilinonaphthalene-8-sulfonic acid (1,8-ANS)
2-anilinonaphthalene-6-sulfonic acid (2,6-ANS)
2-(p-toluidinyl) naphthalene-6-sulfonic acid sodium salt
(2,6-TNS)
2-(N-methylanilino) naphthalen-6-suifonic acid sodium salt
(2,6-MANS)
bi5-ANS (1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid,
dipotassium salt)
1-pyrenesulfonic acid, sodium salt
2-(N-octadecyl) aminonaphthalene-6-sulfonic acid, sodium salt
Table 5--Continued
Cationic Fluorophores
1,1'-dihexadecyloxacarbocyanine, perchlorate (Di-OC.sub.16 (3))
3,3'-diotadecyloxacarboxyanine perchlorate ("DiO", DiOC.sub.18
(3))
1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanine, perchlorate
(DilC.sub.12 (3))
1-1'-dihexadecyl-3,3,3',3'-tetramethyolindocarbocyanine perchlorate
( DilC.sub.16 (3))
1-1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate
(":Dil", DilC.sub.18 (3))
1,1'-didocosanyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate
(DilC.sub.22 (3))
1,1'-dioctadenyl-3,3,3',3'-tetramethylindodicarbocyanine
perchlorate (DilC.sub.18 (5))
3,3'-dioctadecylthiacarbocyanine perchlorate (DiSC.sub.18 (3))
octadecyl rhodamine B, chloride salt (R 18)
rhodamine 6G, octadecyl ester, chloride
rhodamine 101, octadecyl ester, chloride
N-4-(4-didecylaminostyryl)-N-methylpyridinium iodide
(4-di-10-ASP)
1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene,
p-toluenesulfonate (TMA-DPH)
6-palmitoyl-2-(((2-(trimethyl) ammonium)ethyl)methyl-)amino)
naphthalene, chloride (PATMAN)
1-pyrenemethyltrimethylammonium iodide
1-pyrenebutyltrimethylammonium bromide
3-(-anthracene) propyl trimethylammonium bromide
Acridine orange-10-dodecyl bromide (dodecyl acridine orange)
acridine orange-10 nonyl bromide (nonyl acridine orange)
Neutral Fluorophores
1,6-diphenyl-1,3,5-hexatriene (DPH)
1-phenyl-6-((4-trifluoromethyl)phenyl)-1,3,5-hexatriene
(CF-DPH)
palldium disodium alizarinmonosulfonate (Pd(QS).sub.2)
Nile Red or 9-diethylamino-SH-benxo[]phenoxazine-5-one
6-propionyl-2-dimenthylaminonaphthalene (prodan)
6-dodecanoyl-2-dimethylaminonaphthalene (laurodan)
N-phenyl-11-naphthylamine
1,10-bis-(1-pyrene) decane
1,3-bis-(1-pyrene) propane
Table 5--Continued
p-dimethylaminobenzylidenemalononitrile
N-(5-dimethylaminonaphthalene-1-sulfonyl) hexadecylamine
N-(5-dimethylaminonaphthalene-1-sulfonyl) dihexadecylamine
4-(N,N-dihexadecyl) amino-7 nitrobenz-2-oxa=1,3-diazole (NBD
dihexadecylamine)
4-(N,N-dioctyl) amino-7-nitrobenz-2-oxa-1,3-diazole
(NBD-dioctylamine)
4-(hexadecylamino)-7-nitrobenz-2-oxa-1,3-diazole (NBD
hexadecylamine)
1-pyrenecarboxaldehyde
1-pyrenenonanol
7-dimethylamino-4-pentadecylcoumarin
cholesteryl anthracene-9-carboxylate
1-pyrenemethyl 36-hydroxyl-22,23-bisnor-5-cholenate (PMC)
1-pyrenemethyl 38-(cis-9-octadecenoyloxy)-22,23-bisnor-5-cholenate
(PMC oleate)
25-(NBD-methylamino)-27-norcholesterol (NBD-MANC)
25-(NBD-methylamino)-27-norcholesteryl oleate (NBD-MANC oleate)
22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)
amino)-23,24-bisnor-5-cholen-38-9yl
22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)
amino)-23,24-bisnor-5-cholen-38-yl linoleate
N-(3-sulfopropyl)-4-(p-didecylaminostyryl) pyridinium, inner salt
(DilOAS-PS)
3-(N,N-dimethyl-N-(1-pyrenemethyl) ammonium) propanesulfonate,
inner salt
4-(N,N-dimethyl-N-(1-pyrenemethyl) ammonium) butanesulfonate, inner
salt
N-e-(5-dimethylaminoaphthalene-1-sulfonyl)-L-lysine (dansyl
lysine)
Nile Blue
TABLE 6
Polarity-Sensitive Chromophores
Phospholipid Chromophores
2(3-diphenylhexatrienyl) propanoyl-3-palmitoyl-La-a-phosphatidyl
choline (DPH-PC)
N-(6-(biotinoyl) amino hexanoyl)
dipalmitoyl-L-a-phosphatidylethanolamine triethyl ammonium salt
(biotin-X-DPPE)
N-((4-maleimidylmethyl) cyclohexane-1-carbonyl)
dipalmitoyl-L-a-phosphatidylethanolamine triethylammonium salt
(MMCC-DPPE)
N-((2-pyridyldithio) propionyl)
dipalmitoyl-L-a-phosphatidylethanamine triethylammonium salt
Anionic Chromophores
15-phenylpentadecanoic acid
5-(N-hexadecanoyl) amino fluorescein diacetate
The Polymeric Substance:
The thin film forming each semi-selective sensing receptor unit
utilizes a polymeric substance to hold and contain the dye reagent.
The present invention intends that any of the conventionally known
polymers reported in the scientific literature or commercially
available from industrial sources be employed, the particular
choice, chemical composition, specific formulation and state of
preparation being at the discretion of the user.
In this context, two general formats for polymeric substances are
known: a fully prepared polymer or copolymer, existing in bulk as a
polymerized composition; and those reagent materials such as
monomers, co-monomers, cross linkers and the like which are
combined into a reaction mixture and then polymerized in-situ by
any of the conventionally known techniques to yield a polymeric
substance. Either mode of polymeric substance is suitable for use
in making the thin film, semi-selective, sensing receptor unit.
A. Pre-Existing Bulk Polymer Material
The first category of polymeric substance requires that the bulk
polymer material be soluble in one or a mixture of solvents or be
suspendable and carried by one or more solvents. The bulk polymer
is dissolved into and by the chosen solvent (such as chloroform or
toluene ), sometimes with the addition of heat. A fixed amount of
bulk polymer is dissolved in a premeasured aliquot of solvent to
yield a predetermined concentration of dispersed polymeric
solution. The dye reagent of choice (in liquid form) is then
preferably added as a concentrated stock solution to the dissolved
polymer to form the admixture. The prepared admixture of dye
reagent/polymer is then cast, spin coated, or molded into a thin
film having the requisite dimensions; and in a configuration and
size (surface area) to meet the intended application. A number of
conventionally known techniques are available to achieve the
desired result.
A representative listing of previously prepared bulk polymers
suitable for use is given by Table 7 and 8. Also, a list of
conventionally available solvents for dissolving the bulk polymer
prior to admixture with the dye reagent is given by Table 9.
It will be explicitly understood, however, that the lists of Tables
7, 8, and 9 respectively are merely illustrative of those bulk
polymers and organic solvents common to the laboratory and easily
obtainable from commercial sources. The listings are thus
non-inclusive, not exhaustive, and neither limiting or restrictive
to the user; and that any other choice of bulk polymer and organic
or aqueous solvent(s) may be substituted at will or as dictated by
the intended use application.
TABLE 7
Bulk Polymers.
Silicones and Silicon-Containing Polymers:
Polydimerthylsiloxanes
T-structure polymers
Organohydrosiloxane polymers
Polymethylalkylsiloxanes
Fluoroalkylsiloxanes
Aromatic (phenyl containing) siloxanes
Aromatic polymers with functional groups
Aromatic substituted alkyl polysiloxanes
Silicone gums
Polysilanes
Polysilazanes
Polyalkoxysiloxanes-polysilicatse
T-resins and ladder polymers
Silane-modified polymers
Other Polymers:
Polyethylene
Polypropylene
Polymethylmethacrylate
Polystyrene
Polyhydroxyethylmethacrylate
Polyurethanes
Polyvinylchloride
Polyvinylidenc chloride
Fluorinated Polyolefins
Chlorofiuoropolyolefins
Polysubstituted siloxanes
Parafilm
Polyacrylamide
Polyhydroxymethacrylate
TABLE 8
Specific Polymeric Substances.
Poly (2,6-dimethyl-p-phenylene oxide)
Polycaprolactone
Poly (1,4-butylene) adipate
Polyethylene succinate
Phenoxy resin
Polycarbonate
Polysulfone
Polyethylene glycol
Polydimethylsiloxane, trimethylsiloxy terminated
Fluoropolyol
Polyethylenimine
Ethyl cellulose
Polyepichlorohydrin
Poly [bis(cyanoallyl)]-siloxane
Poly (isobutylene)
Polymethylphenyl siloxane
Polyphenyl ether
TABLE 9
Useful Organic Solvents
Acetone;
Chloroform;
Benzene, toluene, phenol and other aromatics;
Alcohols having 1-25 carbon atoms;
Carbon tetrachloride;
Ethers having 2-25 carbon atoms;
Alkanes, alkenes, and alkyenes having from 1-25 carbon atoms;
Thiols having 1-25 carbon atoms;
Organic acids having 1-25 carbon atoms.
Methylene Chloride
Acetone
Ethyl Acetate
B. Polymerizable Thin Film Reagent Mixtures.
When forming and depositing the thin film, it is often desirable
that the dye formulations be combined with monomer compounds to
form a polymerizable reagent mixture. Among the conventional
practices, a variety of different polymerization processes are
known, including thermal techniques, photoinitiated methods,
ionization methods, plasma methods, and electroinitiation
procedures. These different methodologies are exemplified by the
following publications, the text of each being expressly
incorporated by reference herein.
Thermal methods: Graham et al., J. Org. Chem. 44:907 (1979);
Stickler and Meyerhoff, Makromal. Chem. 159:2729 (1978); and Brand
et al., Makromal. Chem. 181:913 (1980).
Ionization methods: A. Chapiro, Radiation Chemistry of Polymer
Systems Chapter IV, Wiley-lntersciences, Inc., New York, 1962a; J.
E. Wilson, Radiation Chemistry of Monomers, Polymers, and Plastics,
chapters 1-5, Marcel Dekker, New York, 1974.
Plasma methods: Yasuda, W. and T. S. Hsu, J. Polym. Sci. Polym.,
Chem. Ed. 15:81 (1977); Tibbet et al., Macromolecules 10:647
(1977).
Electroinitiation methods: Pistoria, G. and O. Bagnarelli, J.
Polym. Sci. Polym. Chem. Ed. 17:1001 (1979); Philips et al., J.
Polym. Sci. Polym. Chem. Ed. 15:1563 (1977); and Odian G.,
Principles of Polymerization, 3rd Edition, Wiley-lnterscience,
Inc., New York.
One preferred method of thin film preparation and deposition is via
the process of thermal polymerization; and employs one or more
temperature activated monomer preparations in admixture with one or
more prechosen light energy absorbing dyes as a polymerizable
formulation [as described in Munkholm et al., Anal. Chem. 58:1427
(1986); and Jordan et al, Anal. Chem. 59:437 (1987)]. Such monomer
preparations typically comprise solutions of several monomers in
admixture and a fixed concentration of at least one light energy
absorbing dye conjugated to an organic carrier which can be
chemically cross-linked. A representative listing of different
monomer compositions suitable for preparing a reaction admixture
which subsequently can be thermally polymerized are given by Table
10; an illustrative listing of conjugated dyes ready for admixture
and photopolymerization is given by Table 10 below.
It will be appreciated that the listings of Table 10 and Table 11
are merely representative of the many different substances which
can be usefully employed in admixture with one or more light energy
absorbing dyes to form the thin film coating.
TABLE 10 ______________________________________ A. Monomers
Acrylamide N,N-methylene bis(acrylamide) (crosslinker)
Hydroxyethylmethacrylate Ethylene glycol
dimethacrylate(crosslinker) Styrene Vinyl acetate
(N-(3-aminopropyl)methacrylamide Hydrochloride [Kodak, Inc.] B.
Comonomer with dimethylsiloxane (Acryloxypropyl)methyl (15-20%)
(Aminopropyl)methyl (3-5%) (Methacryloxypropyl)methyl (2-3%) C.
T-structure polydimethysiloxanes Methacryloxypropyl (25-50%) Vinyl
(50-75%) ______________________________________
TABLE 11
Conjugated dye
Acryloyl fluorescein
acryloyl rhodamine
acryloyl eosin
phenol red
acryloyl 8-hydroxypyrene 1,3 disulfonic acid
acryloyl seminapthorhodafluor
acryloyl seminapthofluorescein
In addition, the scientific and industrial literature provides many
alternative preparations and admixtures which are also suitable for
use in making the present invention; and the dyes may be
incorporated into the thin film by alternative means and techniques
such as entrapment, adsorption, electrostatic binding, and the
like. Accordingly, all of these conventionally known preparations
are considered to be within the scope of the present invention.
VII. The Functions of The Components Comprising Each Thin Film
Sensing Receptor Unit.
A. The Function of the Polymeric Substance:
It is essential to understand the nature, variety, and diversity of
interactions which occur between the dye reagent and the polymeric
substance forming the sensing receptor unit prior to introducing a
sample fluid to the sensor for analysis. Generally, there are two
characteristics for the polymeric material as it relates to the
sensor construction and performance. The first characteristic and
function is the primary role of the polymeric material--capturing
the organic analyte of interest to be detected. This capture
function and capability is performed by absorbing and partitioning
the analyte of interest within the substance and thickness of the
polymeric material itself. The absorption and partition occurs
between the vapor or liquid phase of the fluid sample and the
polymeric material forming one component of the sensor
construction. The partitioning of the analyte of interest may be
similar within the fluid sample and in the polymeric material, that
is the concentration of vapor in each of these two phases may be
the same; or more likely, one of the two will be enriched in
concentration of the analyte relative to the other. Under ideal
circumstances, the polymeric material will serve to concentrate the
analyte of interest via its superior solubility characteristics
relative to the vapor or liquid phase in preferred embodiments of
the receptor units comprising the present invention, the polymeric
material will concentrate the organic analyte, which in turn,
increases the sensitivity and detection limit of the sensor.
The second function and characteristic of the polymeric material,
which will not be present to a similar degree in all embodiments of
the sensing receptor unit, is the spectral influence exerted by the
polymeric material and its ability to alter or modify the spectral
characteristics of the dye independent and separate from the
spectral influences and consequences caused by the analyte of
interest. This second property and characteristic will often vary
with the degree of polarity or the non-polarity of the polymeric
material as individually chosen for use in constructing the
specific embodiment, as well as with the use or non-use of a
solvachromic dye reagent. Polarity as such, however, is not the
sole property or mechanism by which the dye's spectral properties
are mediated or affected. The hydrophobicity/hydrophilicity of the
dye and the polymer together can play major roles; and the
solubility characteristics of the chosen dye within the polymeric
material can also influence the outcome. Thus, the properties of
the polymeric material containing the immobilized dye may or may
not itself alone influence and alter the spectral characteristics
of the immobilized dye apart from and prior to introduction of an
analyte in a fluid sample.
It will be noted, however, that the essential value and
circumstance lies in the polymeric material interacting with the
dye and thus providing a background or baseline interaction and dye
spectral properties against which all other or subsequent optical
determinations and measurements are made and compared. As a
consequence of the dye being contained, dispersed, or otherwise
immobilized within a particular polymeric material, a background or
baseline set of spectral properties for the immobilized and
contained dye is produced which are the result and consequence of
only the interaction between the polymeric material and the dye. It
is this baseline or background set of spectral characteristics
against which all optical determinations and changes in spectral
properties are subsequently made and measured for the detection of
an analyte of interest.
Accordingly, when the sensing receptor unit is then placed in
contact with a fluid sample believed to contain one or more
analytes having or not having an inducible or fixed polarity, the
analytes become captured, absorbed, and partioned by the polymeric
material and generates marked changes in the spectral properties of
the immobilized dye in the sensor. Thus, directly as a result of
the analyte's absorption and partitioning by the polymeric layer,
the spectral light absorbing and light emitting characteristics of
the immobilized dye become changed from its background or baseline
plotted standard provided by the effect of the polymeric material
alone.
B. The Semi-Selective Reaction Function of the Dye Polymer
Reagent.
There is also an essential requirement and function for the dye
reagent in combination with the polymer in each embodiment of the
sensing receptor unit. The admixed dye and polymer combination must
react semi-selectively with the analyte of interest to be detected.
The requirement is easily demonstrated and proven empirically for
any analyte and dye polymer blending via reactive contact; and the
entire series of individual analytes to be identified--whether
introduced singly or as a mixture--must generate a different
spectral response progression for each analyte as a consequence of
reactive contact with that sensing receptor unit. Thus, the
original baseline spectral response yielded by the polymer
substance and dye reagent becomes the internal standard against
which the effects of analyte reactive contact is measured.
In some instances, however, no meaningful modification, alteration,
or change from the original baseline plot will be observed after
reactive contact; and such empirical observation and result proves
an absence of any sensing selectivity (or specificity) of that
individual receptor unit and chemical formulation for that
particular analyte or ligand. It will be noted and appreciated that
not every sensing receptor unit and dye/polymer formulation will or
need be responsive to each and every analyte or ligand presented.
To the contrary, same analytes may cause one or more receptor units
to be quiescent and present baseline signals, while other receptor
units actively respond to yield a spectral response pattern. Under
these circumstances, that chemical constituent combination of dye
reagent and polymeric material may be used to provide a negative
response pattern, but the usage should be employed as an extra
feature in addition to those dye/polymer formulations which show a
positive change in spectral properties.
At the other extreme possibility, two differently formulated
sensing receptor units will empirically exhibit substantially
similar optical signals, spectral response progressions, a
consequence of reactive contact with a single analyte of interest.
The substantial similarity merely duplicates the detection value
and function of having time or spectral plots of optical response.
Thus one of these particular combinations of dye/polymer may be
discarded and replaced for purposes of constructing a heterogeneous
array.
C. Possible Mechanisms of Sensing Receptor Unit Operation and
Function.
The sensing receptor units described herein are not controlled in
operation of function by any particular mechanism of action. The
spectral changes exhibited by the sensors which may be operative,
will include: (1) polarity changes in the polymeric material
generated by the analyte of interest which consequently can impart
changes in the spectral properties of the dyes, as these dyes are
sensitive to polarity; (2) concentration quenching wherein dyes can
associate with the analyte and through this association diminish
their light intensity, the degree of association being influenced
by the presence or absence of the analyte; (3) changes
orientational in nature, in which the polymer, in the presence of
the analyte, orients the dye in a particular way which creates an
environment for changed spectral properties; and (4) swelling in
which the distance between dye molecules changes as a function of
the volume change in the polymeric material caused by the
introduction of the analyte.
VIII. Experiments and Empirical Data
To demonstrate the range and variety of the differently constituted
thin films serving as semi-selective sensing receptor units in an
array as described above, some illustrative experiments were
performed. These experiments and empirical data will serve to
merely demonstrate the utility as well as the diversity of the
membership comprising the array of sensing receptor units of the
present invention. While the experimental design and results are
limited, it will be expressly understood that these empirical
details do not either restrict or limit the membership of the class
in any way. to the contrary, these empirical results and
experiments are merely representative of the number, variety, and
diversity of novel and unique thin films which can be
advantageously prepared and employed in an array and analyzed with
the appropriate computational equipment for the detection of an
analyte of interest.
Experimental Series 1
Materials and Processing
The fluorescent dye, Nile Red (NR), has properties which make it
promising for using as a sensor. The emission (and excitation)
spectrum of the dye, is namely dependent on polarity of its micro
environment. When there is a change in polarity, e.g. by applying a
polar compound, the emission spectrum of the dye shifts and thus
gives either an increase or decrease in fluorescence, dependent on
the detection wavelength and the initial polarity of the micro
environment (=initial position of the emission spectrum). The goal
was to see if, by varying the initial environments of the dye by
incorporating the dye into different organic polymers, polymer-dye
combinations would result which would each respond with a different
change in fluorescence unique for a given analyte.
The first step thus consisted of making thin film combinations of
the fluorescent dye, Nile Red, using a series of different organic
polymers. Then microscope slides with a thin film combination were
analyzed for their spectral responses to several known vapored
chemical compositions. After having found a heterogeneous set of
films which each responded uniquely to the set of known vapor
chemical compositions, the next step was to put the different thin
films in one optical viewing field, so that the films could be
stimulated under the same conditions. To do so, coverslips instead
of slides were coated with the thin film combinations and then
broken into small pieces. Those small pieces were then glued on a
slide with super glue (Dura), which was not fluorescent by
itself.
Silanization
In order to create a good adhesion of the hydrophobic film, the
slides and coverslips, needed to be silanized. First the slides
were cleaned in concentrated sulfuric acid for 15 minutes, after
which they were thoroughly rinsed with distilled water and dried
with acetone. The, freshly made, silanization solution consisted of
95% EtOH (adjusted to pH 4.5-5.5) with 2%
octamethylcyclotetrasiloxane (Petrarch systems) and .was allowed to
stand about 5 minutes, but not much more, before using. The slides
were then agitated in the solution for about 2 minutes, followed by
a brief rinse in 95% EtOH. After drying the slides for 30 minutes
@approximately 60.degree. C., they were ready for use.
Coating of the Slides
In case of the coated slides, all the listed polymers were loaded
with 50 .mu.l of Nile Red (NR) stock solution (2 mg NR/ml
toluene=6.3 mM). The choice of the polymers was based on simplicity
(no polymerization step) and solubility in organic solvents.
The following polymers were tested, followed by the amount
used:
1. Dow corning dispersion coating (DOW), 0.6 g
2. Poly (1,4 butylene adipate) (PBA), Aldrich, 0.5 g
3. Poly (ethylene) succinate (PES), Aldrich, 0.5 g
4. Polydimethylsiloxane, trimethylsiloxy terminated (PDMS), Huls
Petrarch, 1.0 g
5. Poly (2,6 dimethyl) 1,4 phenylene oxide (PDPO), Aldrich, 0.2
g
6. Poly caprolactone (PC), Aldrich, 0.2 g
7. Poly ethylene glycol 8000 (PEG), Sigma, 0.5 g
All polymers were dissolved in chloroform, while heating (oven app.
60.degree. C.), except for DOW and PDMS, which were dissolved in
toluene (no heating required).
Each thin film combination was produced by spin coating the slides.
The thickness of each thin film was influenced by the rotations per
minute and the viscosity of the coating solution. In this case, a
relatively high (unable to be exact) spinning rate and low
viscosity was used, which resulted in a very thin film. Each of the
different thin films dried almost instantaneously. However, the
PDMS film never dried and the PEG film cracked; accordingly, these
films were excluded from the experiments.
For the thin film of the coverslips, the dye content with each of
the polymers was adjusted as necessary so that each thin film
combination would have a similar baseline of fluorescence. The
adjustments were: PES, 15 .mu.l NR stock; and PC, 25 .mu.l NR
stock. The produced thin films were very thin and were estimated to
be less than 5 .mu.m in depth. Each thin film combination showed
the dye reagent to be regularly distributed in the polymeric
substance as a mixture.
Experiment Setup
The experimental instrumentation and apparatus is illustrated by
FIG. 9. Using this system, the testing of the sensors is
accomplished as follows: Vapors are introduced through a vapor
cartridge or they can be introduced directly from a flowing stream
of vapor contaminated air. The air stream is introduced to the end
of the substrate containing the polymer dye combinations. Air is
flowed over the surface for a defined period of time and the
spectral response pattern is observed. Air flow is stopped and the
sensor returns to its baseline signal. The fluorescence changes are
observed by introducing excitation light through a dichroic mirror
onto the substrates. The returning fluorescence is then collected
through an emission filtering scheme and collected by a
two-dimensional detector such as a CCD detector. The resulting
intensity images are collected via computer and stored for
subsequent signal processing.
Via the experimental setup shown in FIG. 9., the computer, in
short, is taking a movie of 64 fluorescence pictures (frames), each
representing 33 milliseconds, separated by an 0.3 second interval.
During the movie, approximately 19 seconds long, a chemical vapor
is puffed on the array for 5 seconds. The change in fluorescence
for each thin film sensing receptor unit (Excitation=530 nm,
Emission=610 nm) is presented on the screen as a change in the
pixel values. The percent change in pixel value as compared to the
first frame, was averaged over the entire film in focus and plotted
against time.
The following chemical compositions in gaseous form were tested:
Air, amyl acetate, benzene, carbon tetrachloride, ethylene
dichloride and toluene. To apply the chemical vapor, the airflow
(approximately 100 ml/min) was led over a filter paper soaked in
the compound (liquid) before the airflow was introduced to the
array of thin films.
Evaluations
An initial evaluation of the thin film sensing receptor units was
performed using the DOW, PDPO, PC, and PES thin films individually.
To demonstrate the semi-selectivity reactivity of each thin film
combination, a series of gaseous chemical compounds were
individually introduced to each thin film. The spectral responses
and the changes in emitted fluorescence (in pixel values) as a
function of time progression (as frames per second) are graphically
plotted for each thin film sensing receptor unit by FIGS. 10-13
respectively. The DOW thin film semi-selective reactivity is shown
by FIGS.: 10A and 10B; the PDPO thin film semi-selective reactors
are revealed by FIGS. 11A and 11B; the PC thin film semi-selective
reactions are demonstrated by FIGS. 12A and 12B; and the PES thin
films' reactions are graphically shown by FIG. 13.
As is shown in FIGS. 10-12, the different thin films on individual
slides DOW, PDPO and PC gave unique responses to the tested
compounds. Moreover, each thin film sensing response was fast and
reproducible. The arrows indicate the on- and off times of the
vapor puff. The second run followed directly on the first run, at
the same spot on the film and with the different chemical vapors
given in the same order: 1 air, 2 amyl acetate, 3 benzene, 4 carbon
tetrachloride, 5 ethylene dichloride and 6 toluene. In contrast as
shown by FIG. 13, the PES thin film showed no response at all (note
the different pixel value scale ), although it did fluoresce
intensely. It will be noted and appreciated that the thin film with
PBA did respond moderately to most odors, but ethyl dichloride
seriously damaged the film. The PBA film was therefore eliminated
from any further testing.
To assess if the different chemical vapors influenced each others
response, the experiment was then repeated, using PC as the model
thin film sensing unit. The order in the second run, however, was
altered. As can be seen in FIGS. 14 and 15, the change of order did
not introduce major differences. Furthermore, this follow up
experiment was performed using another viewing field on the thin
film. When the response of PC thin film to the different vapors is
compared with that in the prior experiment shown by FIGS. 12A and
12B, one can observe it is nearly identical.
FIGS. 15A and 15B also show the effect of vapor puff duration (1,
2, 3, 4, 5 seconds) on the response. This experiment was also
performed on PC thin films using benzene as the gaseous compound.
FIG. 15A shows a duration dependent change in spectral response.
The order of the different spectral responses as indicated by the
legend below the graphs. During the second run shown by FIG. 15B,
the PC thin film seemed to fail responding to various chemical
vapors after the 2 second puff. The PC thin film was probably
exhausted at this stage.
In conclusion from these initial experiments, the four thin films
DOW, PDPO, PC and PES were very promising as a heterogeneous sensor
array, in that they all give distinct, reproducible spectral
response progressions to the tested chemical vapors. The
non-responding PES thin film sensing unit is interesting because it
can be used as an internal control (e.g. to correct for bleaching
and instrument drift).
Experimental Studies
For the subsequent experiments, the individual coverslips holding a
thin film dye and polymer combination were broken into small
pieces; and the small pieces intermixed as an array in the optical
viewer's field. The corrected dye contents of the films turned out
to be successful, in that the baseline fluorescences of the four
thin films were nearly identical. This arranged setting was tested
in the same paradigm as the individually coated slides above; and
again the responses of each thin film sensing receptor unit were
unique and reproducible. This is demonstrated by FIGS. 16-21
respectively.
Experimentally, it was observed that some thin films bleached
during the second trial run (see figures). In our paradigm, the
thin films of the array were exposed for 19 seconds to high
intensity excitation light. This, plus the used low dye content, is
deemed to be the reason for the observed bleaching. Such bleaching
can be diminished by using a shutter which only allows light on the
preparation for the 33 milliseconds a frame is taken.
Experimental Series 2
Another experimental series was undertaken to demonstrate the
feasibility an desirability of using a bundle of optical fiber
strands as the substrate for the semi-selective sensing receptor
units. Although the quantity of empirical data is limited, the
empirical results clearly and unequivocally demonstrate the
operability and the utility of an optical fiber based sensor and
testing methodology.
Optical Fiber Sensor Array Preparation
The polished proximal ends of nineteen 300 .mu.m-diameter fibers
were packed into a stainless steel sheath (1.5".times.0.25"o.d.,
0.125"i.d.) and epoxied in a
twelve-around-six-around-one-formation, with the fiber tips flush
with the end of the sheath. The distal end of each individual fiber
was identified and numerically labeled according to its position
relative to fiber 1 (arbitrarily selected). The distal ends were
then cleaved at even lengths and temporarily bound together (with
heat-shrink tubing) for simultaneous polishing. Fibers were
polished manually in a four-step process using the following
gradient of lapping films: 30, 15, 3, and 0.1 .mu.m. The tubing was
removed and the fibers were soaked in concentrated sulfuric acid
for two hours to clean.
The bundle was separated into two groups for silanization. The
silanizing reagent chosen was based on the nature of the
subsequently deposited polymer overcoat. Group I consisted of
fibers 2, 4, 6, 8, 10, 12, 13, 15, and 17, while Group 2 contained
fibers 1, 3, 5, 7, 9, 11, 14, 16, 18, 19. Group 1 was placed in a
10% solution of (3-trimethoxysilyl) propyl methacrylate 97% in
acetone, in the dark, for two hours. The fibers were then rinsed
with acetone and cured in the dark for one hour. For silanization
of Group 2, a 2% solution of n-octadecyltriethoxy silane in 95%
EtOH adjusted to pH 4.5 with acetic acid was prepared and allowed
to sit for five minutes. Group 2 was agitated in the 2% solution
for two minutes, removed, dipped once in 95% EtOH, and cured at 110
C for 10 minutes.
Polymers
Polymer choices were carried out as follows: fiber 1, PEG(poly
ethylene glycol, from Aldrich); fiber 2, PS078.5 (triethoxysilyl
modified, polybutadiene (50% in toluene) from United Chemical
Technologies, Inc.); fibers 3 and 19, Dow (dimethyl siloxane Dow
dispersion coating, from Dow Corning); fibers 4 and 13, PS078.8
(diethoxymethysilyl-modified polybutadiene in toluene, from United
Chemical Technologies, Inc. ); fiber 5, blank; fibers 6 and 17,
CPS2067 (acryloxypropylmethylcyclosiloxane, from United Chemical
Technologies, Inc.); fibers 7 and 18, PC (polycaprolactone, from
Aldrich); fibers 8 and 15, PS802 (80-85%) dimethyl
(15-20%)-(acryloxypropyl) methylsiloxane copolymer, from United
Chemical Technologies, Inc.); fibers 9 and 14, PBA
(poly(1,4-butylene adipate ) from Aldrich ); fiber 10, PS901.5
(poly(acryloxypropylmethyl siloxane) fibers 11 and 16, PDPO
(poly(2,6-dimethyl-1,4-phenylene oxide), from Aldrich); and fiber
12, PS851 ((97-98%)dimethyl(2-3%)-(methacryxypropyl)methyl siloxane
Co, from United Chemical Technologies, Inc. ).
The two methods used to coat the fiber distal ends were
photopolymerization (Group 1) and dip-coating (Group 2). Monomers
used in photopolymerization were PS078.5, PS078.8, CPS2067, PS802,
PS901.5, and PS851. Dip-coated polymers used were DOW, PEG, PC,
PBA, and PDPO.
Photopolymerization
500 .mu.L of polymer was mixed with 400 .mu.L of Kodak Nile Red dye
solution (1 mg/mL in dichloromethane) and 5 mg of the initiator
benzoin ethyl ether. The steel sheath was then inserted into an X-Y
fiber chuck attached to a deposition system comprised of the
following components: mercury-xenon lamp, neutral density filter,
UV light filter, focusing lens, and pin-hole. The light was focused
onto the proximal end of fiber 2 using the ND filter, and the
distal end of fiber 2 was isolated. The above polymer/dye solution
was drawn up into a capillary tube, dipped onto the distal end of
fiber 2, and exposed to 20 .mu.W of 350 nm UV radiation for 30
seconds.
This process was repeated for the remaining fibers in Group 1, with
different polymers, amounts of BEE, and lengths of UV exposure
time, as listed in Table E1 below.
TABLE E1 ______________________________________ Amount of polymer
Amount of Amount of Dur. of UV Polymer (.mu.L) Nile Red (.mu.L) BEE
(mg) exposure(s) ______________________________________ PS851 500
400 30 30 CPS2067 500 400 0 10 PS078.5 500 400 5 30 PS078.8 500 400
30 60 PS901.5 500 400 0.5 30 PS802 500 400 30 10
______________________________________
Dip Coating
0.48 g of poly ethylene glycol (PEG) was dissolved in 2 mL toluene,
and mixed with 50 .mu.L of Nile Red solution. Fiber 1 was clamped
in a vertical position, distal end facing up. The PEG/Nile Red
solution was drawn up into a capillary tube, carefully dipped onto
fiber 2, and allowed to dry for one minute. This process was
repeated two additional times, resulting in an even layer 70 .mu.m
thick.
The same procedure was performed on the remaining fibers of Group 2
with different polymers (as assigned above), and different
solvents, as shown by Table E2 below:
TABLE E2 ______________________________________ Amt. Amt. Polymer
polym. (g) Solvent solv. (mL) Nile Red (.mu.L)
______________________________________ DOW 0.6244 toluene 2 50 PEG
0.48 chloroform 1 50 PC 0.19 chloroform 2 50 PBA 0.5 chloroform 1
50 PDPO 0.2 chloroform 1.5 50
______________________________________
For identity and convenience purposes, especially with respect to
FIG. 22 and 24-26, a correlation of optical fiber strands and the
corresponding polymer/dye formulated coatings is given by Table E3
below.
TABLE E3 ______________________________________ Optical Polymer/dye
Polymer/dye fiber strand identity no. Optical fiber strand identity
no. ______________________________________ f1 p1 f10 p10 f2 p6 f11
p5a f3 p2a f12 p11 f4 p7a f13 p7b f5 no coating f14 p4b f6 p8a f15
p9a f7 p3b f16 p5b f8 p9b f17 p8b f9 p4a f18 p3a f19 p2
______________________________________
The overall result of the photopolymerization and the dip-coating
processing is illustrated by FIG. 22 which shows an overhead
distal-end view of the prepared sensor comprising individual
optical fiber strands (appearing as f1-f19 respectively). The
bundle of optical fibers 200 is contained by the epoxy filler 204
within the stainless steel sheath 202. The distal end face 206 of
each optical fiber strand (except for fiber 5) is at least
partially covered by a polymer/dye film coating 208 formulated as
previously described herein. The distal end of this prepared bundle
of fibers thus is an array presenting multiple semi-selective
sensing receptor units which differ in their constituent chemical
formulations and which are immobilized on the distal end faces of
the optical fiber strands.
The testing apparatus
The prepared optical fiber sensor array of FIG. 22, comprised of 18
semi-selective sensing receptor units, is positioned in the test
apparatus schematically shown by FIG. 23. It will be recognized and
appreciated that the apparatus of FIG. 23 is a variant of those
previously shown and described by FIGS. 2 and 9 respectively; and
is illustrated in essentially schematic form merely to show the
positioning of the fiber optic bundle with respect to the other
components of the general testing system and instrumentation
already described.
Using the system of FIG. 23, the testing of vapor samples is
achieved as follows: Vaporous samples are introduced to the optical
fiber sensor via computer controlled delivery means to the distal
end of the optical fiber bundle and the individual polymer/dye
coated distal end faces forming the array of semi-selective sensing
receptor units. Air is initially flowed over the optical fiber
array for a defined period of time and the baseline spectral
response pattern is observed. The fluorescence changes are
generated (using the apparatus of FIG. 23) by introducing
excitation light through a dichroic mirror onto the proximal end of
the optical fiber bundle. The excitation light is then carried by
the individual optical fiber strands to the distal end of the
bundle and and excites the 18 thin film coatings of polymer and dye
on the distal-end faces of the array in reactive contact with the
vapor sample. The fluorescent light emissions from each of the 18
polymer/dye coatings on the distal-end faces of the optical fiber
sensor array are then collected through an emission filtering
scheme and collected by a two-dimensional detector such as a CCD
detector. The resulting intensity images are collected via computer
and stored for subsequent signal processing.
Experimental procedure and empirical result
Via the testing setup shown by FIG. 23, a vapor sample containing
air and benzene is puffed onto the optical fiber sensor array of
sensing receptor units for about 5 seconds. The change in
fluorescence from each polymer/dye combination thin film at the end
of each optical fiber is presented as a change in pixel values. The
percent change in emitted light intensity (as measured in pixel
values) for each sensing receptor unit in the optical fiber array
over time (as measured in frames) is plotted graphically and is
shown by FIGS. 24, 25, and 26 respectively.
It will be recognized that FIGS. 24, 25, and 26 illustrate the
changes in fluorescent light intensity for different groupings of
individual sensing receptor units (polymer/dye combinations)in the
optical fiber sensor from the overall total of 18 individual
spectral responses actually generated by the array. FIG. 24 reveals
the spectral responses in light emission intensity yielded by the
array after reaction to benzene for the polymer/dye thin film
coatings identified as p3a, p4a, p5a, p6, p10, and p11 respectively
as a grouped collection pattern. In comparison, FIG. 25 reveals the
grouped collective pattern of spectral responses over time in
reaction to benzene provided by the array using only the
polymer/dye coatings identified as p1, p2a, p2b, p3b, p4b, and p5b
respectively. Lastly, FIG. 26 demonstrates the pattern of spectral
responses generated over time by the array in reaction to benzene
for only the polymer/dye coatings identified as p7a, p7b, p8a, p8b,
p9a, and p9b respectively.
Conclusions
Via the empirical date of FIGS. 24, 25, and 26, it will also be
noted and appreciated that the membership of each group forming the
collective pattern of spectral responses in each instance has been
arbitrarily assigned; and that the user can choose, at will and as
he personally wishes, which among the 18 individual spectral
responses to use in creating a group or collective spectral
recognition pattern by which to identify and distinguish benzene
from other similar and dissimilar chemical compounds. In effect,
the user may use less than six spectral response to form a spectral
recognition pattern from among the 18 spectral responses actually
available; or may use any six spectral response progressions among
the 18 actually available to form a grouped pattern (as shown by
FIGS. 24-26); or may use more than six spectral responses to form a
collective recognition pattern; or may (as in distinguishing among
homologs of the same chemical compound series) use all 18 spectral
responses generated by the optical fiber sensor to form one single
spectral recognition pattern.
The present invention is not to be limited in scope nor limited in
form except by the claims appended hereto.
* * * * *